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9A0-094 - Adobe(R) Photoshop CS4 ACE - Dump Information

Vendor : ADOBE
Exam Code : 9A0-094
Exam Name : Adobe(R) Photoshop CS4 ACE
Questions and Answers : 123 Q & A
Updated On : April 24, 2019
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9A0-094 Adobe(R) Photoshop CS4 ACE

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9A0-094 exam Dumps Source : Adobe(R) Photoshop CS4 ACE

Test Code : 9A0-094
Test Name : Adobe(R) Photoshop CS4 ACE
Vendor Name : ADOBE
Q&A : 123 Real Questions

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ADOBE Adobe(R) Photoshop CS4 ACE

Adobe Unveils creative Suite CS4, GPU Accelerated Photoshop | killexams.com Real Questions and Pass4sure dumps

The embargo on Adobe's creative Suite 4 lifted tonight at midnight and a number of information articles have revealed the details of the newest update to Adobe's flagship application package.

Most interestingly, CNet experiences that rumors that Adobe's Photoshop CS4 would employ the use of GPU-accelerated graphics were correct.

The new edition of Adobe's flagship software product takes its first steps in using the photographs processing unit, or GPU, said John Nack, predominant product manager for Adobe Photoshop. as an example, the photographs chip helps Photoshop CS4 fluidly zoom in and out, rotate the canvas so artists can reorient a picture for the ultimate sketching angle, reveal and manipulate 3D objects, and handle color correction.

despite the fact, one GPU function did not rather make the reduce for CS4 is known as "Pixel Blender" which enables users to rapidly observe special outcomes. in line with Nack, the characteristic is often a free update from Adobe Labs.

a couple of of the different new Photoshop CS4 aspects are highlighted at Macworld together with:

- content-mindful scaling- Nondestructive corrections within the Adjustment panel- improved Lightroom integration- advanced three-D points in Photoshop extended- New Tabbed interface

Adobe Photoshop CS4 will cost $699 and ship in October. current Photoshop CS3, Photoshop CS2, or Photoshop CS homeowners can improve for $199. Adobe Photoshop prolonged will charge $999 and present Photoshop CS3 prolonged, Photoshop CS2 or Photoshop CS owners can upgrade for $349.

Of route, Adobe also brought CS4 versions of their different functions. further coverage:

Photoshop CS4: TGDaily First seem to be, "Sneak Peek" VideosIndesign CS4: MacworldIllustrator CS4: CNet, Macworld, DigitalArtsOnlineAfter consequences CS4: DigitalArtsOnlinePremiere pro CS4: DigitalArtsOnlineSoundBooth CS4: CNet

Full details and pricing tips can also be acquired from Adobe.com.

Adobe is selling CS4 programs via their online store or Amazon.com.

Adobe CS4 grasp collection – download Free 30-Day Trial | killexams.com Real Questions and Pass4sure dumps

adobe cs4 trial

adobe cs4 trial

The wait is over. you could now down load a 30 day trial of all Adobe application that are part of the Adobe artistic Suite four household.

This includes Photoshop CS4, Dreamweaver CS4, Illustrator CS4, superior seasoned CS4, SoundBooth CS4, Indesign CS4 and After effects CS4.

many of these installers applications are well over a GB in size so when you are on a gradual connection, a much better thought can be ask Adobe for the CS4 DVD (master collection) that charges round $15.

These are full functional utility and you can convert the trial version right into a retail edition by way of effortlessly coming into the serial quantity and activating over the information superhighway or mobile.

and you can legally installation Adobe CS4 on two computers the use of a single license.


Adobe launches Photoshop-Lightroom subscription | killexams.com Real Questions and Pass4sure dumps

Adobe wants photography pros and enthusiasts to sign up for a subscription that bundles Lightroom, Photoshop, and some services for $10 a month. It's only for those who bought a license to CS3 or later, and the $10 price is a limited-time offer through the end of 2013.Adobe desires photography execs and fans to check in for a subscription that bundles Lightroom, Photoshop, and a few features for $10 a month. it's handiest for those who bought a license to CS3 or later, and the $10 expense is a restricted-time offer during the end of 2013. Adobe programs

Adobe techniques on Tuesday introduced the availability of its $10-per-month subscription for Lightroom and Photoshop and launched Lightroom 5.2, which expands and refines the utility's picture-enhancing tools.

more particulars on Lightroom 5.2 are beneath, however here's how the brand new subscription works. it be known as the Photoshop images program, and or not it's accessible best to those who have a license to Photoshop CS3 or later. not like the discounted promotional fee for its broader inventive Cloud subscription, which charges creative Suite shoppers $30 per month for the first year after which $50 in a while, the Photoshop-Lightroom subscription cost does not enhance. however valued clientele must register by means of the conclusion of 2013.

The subscription additionally comprises membership in the the Behance sharing site, a sort of social community for creative individuals who wish to publicize their work online, online practising components, and 20GB of synchronized online storage. Adobe offered the program in keeping with criticism that its full-fledged creative Cloud program is just too expensive for photography fans. however Adobe has mostly ceased updating its CS version of Photoshop bought via perpetual licensing, it continues to promote Lightroom licenses for about $one hundred fifty.

no longer all and sundry is happy with the subscription program. "nowadays I spoke to a direct income rep, and he informed me that i used to be no longer eligible as a result of I selected to get the total CS6 suite during the artistic Cloud in 2012 and that my academic discount types (CS4 and CS5) do not qualify me," one client calling himself George complained in a remark to Adobe's announcement. "despite the fact that I can't get the $9.ninety nine per month rate, I feel I should be in a position to get each Photoshop and Lightroom within the single-app bundle for $19.ninety nine per month."

one after the other, Adobe talked about purchasers are signing up for the total artistic Cloud subscription faster than expected, with more than 1 million subscribers as of August 30.

The subscription program has angered some valued clientele who do not are looking to use application that stops working in the event that they cease paying and who say Adobe is elevating fees. Adobe argues that the application means purchasers will get access to a broader latitude of application, get updates sooner, and faucet into new online features. buyers rewarded the business through pushing its inventory rate up in accordance with the subscription numbers.

David Wadhwani, the chief of Adobe's creative application products, tried to convince skeptics that the inventive Cloud will extend to present more value sooner or later.

"inventive Cloud continues to be in its infancy, and we now have much more to do," Wadhwani spoke of a blog publish. "And thanks partially to your feedback, the yr ahead may still be fun and wonderful as we continue...to construct a inventive platform with fully built-in software, capabilities and hardware."

separately, Adobe announced its foray into hardware with its Mighty stylus, which it'll start promoting within the first half of 2014 in cooperation with stylus maker Adonit. The stylus taps into some inventive Cloud features, for instance letting an artist reproduction art to a digital clipboard after which paste it into a coworker's project the usage of the pen.

The Adobe Mighty stylusThe Adobe Mighty stylusThe Adobe Mighty stylus Adobe programs

Lightroom gets "smoothness" adjustmentAdobe additionally launched Lightroom 5.2, the primary predominant update to the more recent version of the photo modifying and cataloging software. There turned into no Lightroom 5.1 on account of timing complications involving the unencumber of Lightroom and Photoshop, which share the same engine for processing raw-structure photographs.

Lightroom 5.2 adds a brand new modifying tool, a slider called "smoothness" this is designed to cut back color-mottling artifacts which are most obtrusive in digital pictures taken at high sensitivity, high-noise settings. These mottled shades frequently reveal up as gigantic blotches of reddish or greenish tint to the image colours.

Adobe also introduced improvements to the spot-healing device, which is used to do issues like remove dermis wrinkles or filth spots, as an instance by letting photographers characteristic the fringe of the enviornment being edited. And the auto exposure algorithm has been better for people that need to let Lightroom take a first flow at picture enhancing settings, Adobe referred to.

additionally new in Lightroom 5.2 is aid for the Canon 70D, an SLR prone to be prevalent with photo enthusiasts. other new cameras supported:

• Canon PowerShot G16

• Casio Exilim EX-ZR800

• Fujifilm FinePix HS22EXR

• Fujifilm FinePix HS35EXR

• Fujifilm FinePix S205EXR

• Fujifilm X-A1

• Fujifilm X-M1

• Leica C (Typ 112)

• Panasonic Lumix DMC-GX7

• Panasonic Lumix DMC-FZ70

• Panasonic Lumix DMC-FZ72

• Pentax Q7

• Pentax okay-50

• Pentax ok-500

• Sony DSC-RX100 II

• Sony A3000 (ILCE-3000)

• Sony NEX-5T

Lightroom additionally includes preliminary help for two very new cameras, the Canon PowerShot S120 and Olympus OM-D E-M1. Preliminary help for phase One's medium-format IQ260 that was within the beta version changed into taken off the listing, although.

for those who shoot tethered -- during which a USB cable pipes images at once into Lightroom from the digital camera as they're shot -- Lightroom 5.2 now supports the Canon EOS 6D, the Canon revolt rebellion T5i / 700D, the Canon EOS rebel SL1 / EOS 100D, and the Nikon D7100.

For lens corrections, Lightroom 5.2 can repair photos from the GoPro Hero three and a number of Hasselblad, Sony, and Sigma lenses.

Adobe Creative Cloud graphicAdobe Creative Cloud graphicAdobe programs is attempting to circulate its shoppers to its creative Cloud subscription. Adobe programs

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Adobe(R) Photoshop CS4 ACE

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Five Adobe CS4 goodies for photographers | killexams.com real questions and Pass4sure dumps

As you might expect, Adobe’s $699 Adobe Photoshop CS4 ( ) contains a treasure trove of goodies for pixel-pushing photographers. But you’ll also find some unexpected helpers tucked inside CS4’s bundled applications, Adobe Bridge CS4 and Adobe Camera Raw 5. Here are five favorites all photographers should check out.

1. Bridge’s Review Mode

Say you have a folder full of images that you want to winnow down to a collection of favorites. Bridge’s Review Mode is perfect for sorting your images into keepers and tossers. Open Bridge, click on the folder you want to sort, and then press command-B. In this mode, your photos appear as if they’re on a carousel.

Think of this as musical chairs for your photographs. Use the right- and left-arrow keys to navigate from shot to shot. Remove images from the carousel by pressing the down-arrow key. (The photo isn’t deleted from its location in the folder, only removed from the carousel.) Cycle through the shots as many times as necessary until you’re pleased with the survivors. Once you’re satisfied, click on the Make Collection icon in the lower right corner of Review Mode. Bridge gathers your favorites into a virtual collection. Name the collection, and you’re set.

2. Bridge’s Collections

Another of my favorite Bridge CS4 features is the ability to make Collections. These ad-hoc folders are almost identical to that same great feature in Adobe Photoshop Lightroom and to Albums in Apple’s Aperture. Collections let you gather together images in virtual folders without moving the files in the Finder.

Why is this important? Because you want the flexibility to play with your photographs without compromising the organizational structure you use to keep track of them. For example, if you need a folder of cat portraits for a project, create a Collection by clicking on the New Collection button at the bottom of the Collections panel, and then drag in virtual copies of the images from the Content panel or the Finder. When you’re done with the project, you can trash the Collection (click on its name and then click on the trash icon), leaving the original shots untouched and organized according to your folder hierarchy and naming conventions. Bridge collectionsBridge's collections (shown in the Collections panel) let you pull together virtual gatherings of images without changing the organization of your folders.

Even better, Smart Collections automatically assemble photos for you based on a variety of criteria. Want a folder of your best shots? Make a Smart Collection that gathers images you’ve given a rating of 4 stars or higher. To do so, click on the New Smart Collection button at the bottom of the Collections panel, specify your criteria, and then click on Save.

You’ll find the Collections panel on the left side of the Bridge interface. Manually created Collections are represented by brown folders; Smart Collections are blue. (If you don’t see them, click on the Collections tab to reveal them, or select Window: Collections Panel.) Icons for editing Collections, creating new ones, or trashing existing ones are located at the bottom of the Collections panel.

Tip: Need a closer look at an image in a Collection? Click on the image once and then press the spacebar to make the photo fill your screen. Hit the spacebar again to return it to thumbnail size.

3. Adobe Camera Raw’s Graduated Filter

Now that you’ve collected all your best shots, it’s time to learn about one of the most useful new tools in Adobe Camera Raw 5—the Graduated Filter. This feature solves the age-old problem of balancing bright skies and dark foregrounds in your landscape scenes.

Select an image in Bridge and press command-R to open it in Camera Raw. The edits you make here are nondestructive, which means you can play with photos all you want and then return to the original by choosing Camera Raw Defaults in Bridge (select Edit: Develop Settings: Camera Raw Defaults). The Graduated Filter tool is in the top toolbar, fifth from the right. Click on it (or just press G), and then drag the cursor from top to bottom in your image (or any other direction you want). Adobe Camera Raw builds a filter that goes gradually from dark to light, making it perfect for darkening a bright sky without affecting the foreground.

Before and after camera raw's graduated filterAdobe Camera Raw's Graduated Filter tool lets you darken skies without changing the foreground. So instead of the original's less-than-vibrant sky and washed-out water, you'll have a more color-balanced image.

But the Graduated Filter tool isn’t just for exposure adjustments. Once you’ve made your selection, you can also play with sliders for Brightness, Contrast, Saturation, Clarity, Sharpness, and Color. Camera Raw provides you with presets for each of these filters as well, represented by minus (-) and plus-signs (+) at the edges of each slider. In the Graduated Filter’s Color presets, the plus-sign slider applies warm colors (yellow/orange) to the image while the minus-sign slider applies cool colors (blue).

You can apply multiple Graduated Filters to one image, edit them, and even revisit the image later on if you’ve changed your mind.

4. Photoshop’s Targeted Adjustment Tool

Photoshop’s Targeted Adjustment Tool lets you adjust a specific tone or color by clicking and dragging on your image. You’ll find it in both the Curves and Hue/Saturation panels within the Photoshop CS4’s new Adjustments panel (Window: Adjustments).

For example, in the Curves Adjustments panel, click on the hand icon to activate the Targeted Adjustment Tool. Then click on the area of the image you want to adjust and drag up or down to lighten or darken the selected tones. The more you drag, the more intense the adjustment. I usually adjust a light area of the image first and then create a second adjustment in a darker area.

Curves adjustment panelPhotoshop's new Targeted Adjustment Tool makes it easier than ever to work with curves.

You have similar capability in the Hue/Saturation Adjustments panel. To increase Saturation, click on the hand icon and then click and drag on your image to the right. Decrease saturation by dragging to the left. Hold down the command key while dragging to adjust the Hue. Assess the impact of your adjustments by clicking on the eyeball icon in the Layers panel to hide or display the change.

The beauty of this approach is that Photoshop CS4 automatically creates an adjustment layer when you select a tool in the Adjustments panel. Save your file as a layered document to go back at any time and make changes.

Tip: Select File: Save As to save the Photoshop version of your photo, and then place it in the same folder as the original. That way you can view them side-by-side in Bridge.

5. Photoshop’s Effortless Navigation

When you’re working on a large image, it’s easy to get lost in the pixels while making adjustments. While it’s often necessary to work at 100-percent view, that makes it difficult to maneuver from one part of your image to another. Photoshop CS4 has improved its built-in navigation system so you never have to ask for directions.

Now when you’re viewing your image close up and need to move to another part of it, select the zoom tool and then press the H key while holding down the mouse button. Your photo zooms down to Fit In View mode and Photoshop converts the cursor to a rectangle that you can move to any part of the picture. When you let go of the mouse button, Photoshop takes you to the new location but returns you to your magnified view.

Photoshop's Bird's Eye ViewMove around your photo effortlessly with Bird's Eye View, new in Photoshop CS4.

Put Them All Together

The best thing about these new CS4 tools is that you can string them together to create an efficient mini-workflow. They make it that much easier for photographers to select, sort, correct, and view images.

[Senior Contributor Derrick Story is the author of The Photoshop CS4 Companion for Photographers (O’Reilly Media, 2008). Tune in to his weekly podcast The Digital Story.]

To comment on this article and other Macworld content, visit our Facebook page or our Twitter feed.

Simultaneous defeat of MCF7 and MDA-MB-231 resistances by a hypericin PDT–tamoxifen hybrid therapy | killexams.com real questions and Pass4sure dumps

Chemicals and reagents

RPMI 1640 without phenol red, l-Glutamine, penicillin/streptomycin, trypsin, dimethylsulfoxide (DMSO), N-desmethyltamoxifen (NDMTAM), 4-hydroxytamoxifen (4-OHT), tamoxifen HCl (TAM), rotenone (ROT), antimycin-A (ANTI-A), oligomycin (OLIGO), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), polyethylene glycol sorbitan monolaurate (TWEEN 20), bovine serum albumin (BSA), Triton X-100, thiazolyl blue tetrazolium bromide (MTT), 123 bp DNA Ladder, DNA loading buffer, crystal violet, bafilomycin A1, terminal transferase (TdT kit), biotin-16-dUTP, anti-γ-tubulin and sodium pyruvate were purchased from Sigma-Aldrich Norway AS (Oslo, Norway). ProLong® Gold Antifade Reagent with/without DAPI, Image-iT® FX Signal Enhancer, BlockAid™ blocking solution, Quant-iT™ PicoGreen® dsDNA Reagent, LIVE/DEAD™, dithiothreitol, TopVision agarose, 50× TAE electrophoresis buffer, Streptavidin-Alexa Fluor™ 488 conjugate, Hoechst 33258 pentahydrate, MitoTracker® Deep Red FM, and Click-iT® lipid peroxidation imaging kit-Alexa fluor® 488 were purchased from Thermofisher Scientific (Waltham, MA, USA). GelRed™ was obtained by Biotium Inc. (Hayward, CA, USA). All consumables related to a Seahorse XFe96 analyzer were purchased from Seahorse Biosciences Europe (Copenhagen, Denmark). Hypericin (HYP, 99.3%) was obtained from Planta Natural Products GmbH (Vienna, Austria). 17β-Estradiol (E2) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA), Cremophor® EL was obtained from BASF SE (Limburgerhof, Germany), Annexin V-FITC-conjugated was purchased from ImmunoTools GmbH (Friesoythe, Germany), and N-desmethyltamoxifen FITC (NDMTAM-FITC) prepared as described elsewhere24,57 was kindly donated by Dr. K. Yannakopoulou, INN, NCSR “Demokritos”.

Cell culture

For the purposes of this study we chose MCF7 and MDA-MB-231 (triple negative) human breast adenocarcinoma cell lines. All cells were originally obtained from ATCC and grown in RPMI 1640 without phenol red, supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin at 37 °C in a 5% CO2 and 95% humidified atmosphere. Cells were inoculated into 96-well plates (2 × 104 cells/100 μL media/well), six-well plates (1 × 106 cells/2 mL media/well), glass bottom 35 mm Petri dishes (Mattek Corp., 1 × 105 cells/2 mL media/dish) 24 h prior to treatment or confocal microscopy imaging.

Live cell deconvolution imaging

MCF7 and MDA-MB-231 cells were seeded on glass bottom 35 mm dishes (Mattek Corp., 1 × 105 cells per dish) 24 h prior to the experiments. The cells were subsequently treated with (i) 10 μM NDMTAM-FITC 4 h and (ii) 2 μM HYP 4 h. The specific subcellular organelle fluorescent probe for mitochondria (Mitotracker®-Deep Red), was always added to the cells 20 min prior to imaging at a concentration of 150 nM. Cells were subsequently washed with phosphate-buffered saline (PBS) and imaged in fresh 10% fetal calf serum containing RPMI 1640.

Imaging was performed on an OMX V4 instrument equipped with sCMOS cameras and a solid-state light source (GE Healthcare). The system was operated in widefield mode to minimize light dose. Z-stacks covering the whole cell (Z-spacing 125 nm) were acquired. Images were deconvolved and aligned using Softworx software (GE Healthcare).

Confocal microscopy

Cells were seeded on glass bottom 35 mm Petri dishes (1 × 105 cells per dish) 24 h prior to the experiments. After treatment, fixation, permeabilization and labeling as per the Click-iT® lipid peroxidation imaging kit protocol, the cells were treated with DAPI-containing Prolong® Gold antifade reagent (Life Technologies Inc.). The 4-OHT treatment was in this case performed overnight instead of after irradiation, as the cells were fixed 1 h following irradiation and hence 4 h incubation with 4-OHT post-irradiation was not possible.

The cells were examined with a Zeiss LSM 710 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) equipped with a multiline Ar-Laser (458/488/514nm), a DPSS-561 10 (561 nm), a Laser diode 405-30 CW (405 nm), and an HeNe laser (633 nm). Fluorescence images were taken by a Zeiss plan-Apochromat ×63 NA/1.4 oil-immersion. Intensity was measured as described in our previous work58 using Fiji 2.0 v1.51k and Igor Pro 6.36. Briefly regions of interest were determined using a variance filter (kernel 8 pixels), followed by a binary threshold and the particles analyze routine from Fiji. Image processing was performed with Photoshop CS4 (Adobe, Mountain View, CA).

Phototoxicity studies and cell irradiation Treatment and irradiation

Cells were inoculated (20 × 103) into 96-well plates and left to incubate in complete media containing 10% FBS for 24 h. Cells were then treated with media only, 4-OHT (15 μM, post-irradiation unless otherwise specified), HYP (2 μM, 4 h) and HYPERTAM (HYP and 4-OHT). The DMSO content was at all times kept ≤0.25%. Following cell incubation with HYP for 4 h, all treatment groups were washed twice. The cells were irradiated from the plate underside by means of a Lumisource lamp (PCI Biotech AS, Oslo, Norway) through a 530 nm cut-off longpass filter (Roscolab Ltd, London, UK), at an irradiance of 4 mW/cm2. In the combination treatment experiments, 4-OHT (15 μM) was added again to all appropriate cell groups after irradiation.

Cytotoxicity assessment

The redox ability of all cell groups and dark controls was assessed by the MTT assay 24 or 48 h post-irradiation. At these time points the MTT assay outcome was utilized to estimate cell viability. The assay was performed by replacing cell media with complete media containing 0.5 mg/mL MTT and incubating at 37 °C in a 5% CO2 humidified atmosphere for 3 h. MTT media were subsequently aspirated from all cells and the produced formazan crystals solubilized with 100 μL DMSO per well. The plates were shaken for 10 min at ~300 r.p.m. in a Heidolph Titramax 101 orbital shaker (Heidolph Instruments GmbH & Co.KG), and the endpoint absorbance measurements at 570 nm were performed in a BioTek PowerWave XS2 plate reader (BioTek Instruments, Inc.). Blank values measured in wells with DMSO and no cells were in all cases subtracted.

LDH leakage assay

Cells seeded in 35 mm Petri dishes (5 × 105) were left to incubate in complete media containing 10% FBS for 24 h. The cells were subsequently incubated with HYP (2 μM) for 4 h washed, placed in OPTIMEM (Life technologies, 2 mL/dish), irradiated, and then 15 μM 4-OHT was added post-irradiation to the combination treatment groups. At 6 h after treatment, cell media (supernatants) were removed from all cell groups and placed on ice. The media-control cells were further washed with PBS, trypsinized, and centrifuged. The resulting cell pellet was resuspended in 2 mL OPTIMEM containing 0.25% Triton X-100 and agitated for 30 s. The resulting suspension constituted the 100% death control. An assay reaction mixture (pH = 7.4) was prepared containing 100 mM Tris-HCl, 0.4 mg/mL NADH, and 20 mM sodium pyruvate. Seven hundred microliters of the mix were added to 700 μL of sample (all collected supernatants; media-control cell lysate which signified 100% death; OPTIMEM only control) in a 1 mL cuvette with 1 cm light path length. The decay absorbance kinetics of NADH oxidation to NAD+ through pyruvate conversion to L-lactic acid by LDH were in each case monitored at 340 nm (εNADH 340nm = 0.00622 L μmol−1 cm−1), using a Shimadzu UV-2550 UV-VIS spectrophotometer (Shimadzu Corp.).

Clonogenic assays

Four thousand cells were inoculated per 35 mm Petri dish (three dishes per treatment group) and 24 h later were treated with the PDT and 4-OHT protocols as described above. 4-OHT was removed from all relevant treatment groups 24 h post-irradiation and fresh media were added to all treatment groups. Approximately 2 weeks later (or when the colonies were optimal), the cells were fixed with ethanol for 10 min, and stained with crystal violet (0.05%). The cells were washed and the stained colonies containing more than 50 cells were counted in each group.

ER knockdown

105 cells/well were seeded in 12-well dish for transfection the next day. Pools of the following Silencer Select siRNAs were used (Invitrogen) s303873, n303876, s4826, s4827; 20 pmole each, with RNAiMax transfection agent (invitrogen), following the manufaturer’s instructions.

Immunoblots

Cells (~106) were harvested at 6 h following irradiation of groups treated with vehicle, 4-OHT (post-irradiation, 15 μM, 4 h), HYP-PDT (2 μM, 4 h), or 4-OHT (post-irradiation) + HYP-PDT. The lysates were run on NuPage Bis Tris (LC3B) or Novex Tris Gly (ESR) gels. The proteins were subsequently transferred from the gels onto PVDF (LC3B) or nitrocellulose (ESR) membranes, using a Trans-Blot® Turbo™ transfer system (Bio-Rad Laboratories Inc.) (LC3B) or 50 mM Tris, 380 mM Gly, 0.1% SDS, 20% EtOH (ESR). Antibodies used were LC3B 1:2000 (CST 2775S), ESR2 1:1000 (Invitrogen PA1-311), ESR1 1:1000 (MA3-310), γ-Tubulin 1:20,000 (Sigma T6557). Signals were detected using a ChemiDoc™ MP Imaging System and intensities were quantified using Image Lab software (Bio-Rad Laboratories Inc.).

Lipid peroxidation assay

We performed lipid peroxidation investigations using the Click-iT® lipid peroxidation detection with linoleamide alkyne (LAA; Life technologies Inc.). In brief the cells were co-incubated with 50 μM LAA and 4-OHT (overnight the previous night, 15 μM), HYP (2 μM), or 4-OHT overnight + HYP for 4 h, irradiated, and left for 1 h. LAA oxidation led to alkyne modifications at the nucleophilic side chains of proteins. These proteins could then be labeled by Alexa Fluor 488 azide, employing copper-catalyzed click chemistry upon cell fixation (4% formaldehyde, 15 min) and permeabilization (Triton X-100 0.5%, 10 min). Consequently, the cells were studied by confocal fluorescence microscopy (vide supra).

Metabolic studies

In order to study the changes in cell metabolism under the insult of 4-OHT (15 μM), HYP (2 μM), and combination (4-OHT + HYP) with and without irradiation, we employed the Seahorse XFe96 analyzer (Seahorse Bioscience, Copenhagen, Denmark). Cells treated with 4-OHT (overnight), HYP (3h), or combination, had their media changed to XF base medium minimal DMEM (Seahorse Bioscience) supplemented with 10 mM glucose, 2 mM l-glutamine, 2 mM sodium pyruvate and adjusted to pH 7.4. 4-OHT and HYP were maintained in the appropriate groups and the cells were incubated for an extra hour at 37 °C, 0% CO2 (total 4 h). The appropriate cell groups were then irradiated while an identical plate was used as dark control. The basal oxygen consumption and media acidification rates (OCR and ECAR respectively) of the cell groups were measured in four cycles. The four corner wells were left devoid of cells and were used as blank values. These were automatically subtracted by the Seahorse analyser software.

All values obtained were normalized per cell line according to the DNA content. After the Seahorse measurements 25 μL of Quant-iT™ PicoGreen ® was added to 6 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Seahorse media were aspirated and replaced with 50 μL of picogreen-TE buffer. The cells were incubated for 30 min at 37 °C, 5% CO2 and the endpoint fluorescence measurements were read at a BioTek Synergy 2 microplate reader (λex = 480 nm, λem= 525 nm). Blank values were obtained from wells with no cells yet treated with picogreen-TE and were subtracted from all cell group values.

Flow cytometry analyses for apoptosis

MCF7 and MDA-MB cells 231 were seeded in 60 mm Petri dishes (6 × 105 per dish) and left overnight at 37 °C and 5% CO2 humidified atmosphere. The cells were subsequently incubated with media only or HYP (2 μM, 4h). The cells were then washed twice with PBS and then the HYP-PDT and HYPERTAM groups were irradiated with the Lumisource lamp as detailed earlier. 4-OHT was subsequently added to the 4-OHT and HYPERTAM groups (15 μM). After 3, 8, and 24 h the cells were then subjected to two different apoptotic flow cytometry analyses for (A) Annexin V and (B) the TUNEL assay (8 and 24 h).

  • A.

    For Annexin V staining the cells were at the three selected time points trypsinized and stained for 30 min with Annexin V-FITC (5 μL/mL) and LIVE/DEAD IR (1 μL/mL) as per the manufacturers’ instructions. The cells were next washed with and resuspended in PBS containing Ca2+ and Mg2+. The flow cytometric analyses were performed using a CytoFlex S (Beckman Coulter, Brea, CA, USA) on a total of 20,000 events for each sample. FITC fluorescence was excited at 488 nm and registered after a bandpass filter at 525/40 nm. LIVE/DEAD IR fluorescence was excited at 638 nm and emission was collected after a 780/60 nm bandpass filter.

  • B.

    For the TUNEL (terminal deoxynucleotidyl transferase biotin dUTP nick end labeling) assay, the cells were initially trypsinized at the selected time points, and fixed with ice cold (−20 °C) methanol for at least 1 h (100%, 1 mL). Subsequently the cells were incubated in a TdT reaction mixture (TdT enzyme 0.2 μL, TdT reaction buffer 5 μL, CoCl2 3 μL, biotin-16-dUTP 0.5 μL, and DTT 0.5 μL of 10 mM aqueous stock, H2O 40.8 μL—30 min at 37 °C), centrifuged (500 × g, 5 min), supernatant removed and pellets resuspended and incubated in 4% dry milk suspension containing streptavidin-AlexaFluor 488 (1:50 dilution, 0 °C, 30 min). cells were again centrifuged and resuspended in PBS containing Hoechst 33258 (1.5 μg/mL) for 30 min, RT. The flow cytometric analyses were performed using a LSRII (BD Biosciences, Franklin Lakes, NJ, USA) until at least 10,000 events were reached for each sample. AlexaFluor 488 fluorescence was excited at 488 nm and registered after a longpass filter with a cut-on at 505 nm and a bandpass filter at 530/30 nm. Hoechst 33258 was excited at 355 nm and the emission was collected after a 450/50 nm bandpass filter.

  • All flow cytometry data were analyzed using the FlowJo v.7.6.1 software (Treestar Inc., Ashland, OR, USA).

    Animal studies

    For the HYPERTAM in vivo pilot study, inhouse bred, female NOD-scid Il2rγnull (NSG) immunocompromised mice were employed. The mice were inoculated subcutaneously (i.p.) with 7.5 × 105 cells of either MCF7 or MDA-MB-231, suspended in equal volumes of PBS and Matrigel, when they reached 6 weeks of age. After approximately 2 weeks for MDA-MB-231 and 3 weeks for MCF7, the tumor volume reached ~100 mm3, at which time the experiments could commence. The tumor volumes (mm3) were calculated with the help of the following formula: \({\mathrm {tumour}}\,{\mathrm {volume}} = \frac{{({\mathrm {longer}}\,{\mathrm {dimension}}) \times ({\mathrm {smaller}\,{\mathrm {dimension}}})^2}}{2}\) following caliper measurements twice weekly and reported as percentage change after treatment. When volume exceeded 1000 mm3, or mice showed clinical signs of cachexia and/or weight loss more than 20%, they were sacrificed by cervical dislocation under general anesthesia. Animals were allowed to eat and drink water ad libidum, while for the estrogen-dependent MCF7 tumors, the water was supplemented with estradiol (E2) at 6.25 μg/mL.

    For the animal studies six groups of mice were used: control mice (CTRL) that were not administered any drugs or subjected to irradiation, HYP dark (HD) mice which were administered a full dose of HYP but were not exposed to light, TAM only (TAM) which were administered a full tamoxifen dose, HYP and TAM dark (HTD) which were administered full HYP and TAM doses but not light, HYP-PDT group (HL) which were administered PDT and then exposed to light 2 h later, and finally HYP-PDT + TAM which were administered HYP, exposed to light 2 h later, and then administered TAM. Each mouse group contained at least five mice while the groups of particular interest (HL and HTL) contained either six or seven mice.

    From preliminary toxicology and PDT studies (data not shown), the optimal treatment regime was determined as follows: 12.5 mg/kg HYP was administered i.p., 2 h prior to irradiation (40 J/cm2) with the PDT lamp. Following PDT, the HTD, TAM and HTL groups were also dispensed one i.p. bolus of 200 mg/kg TAM. Both HYP and TAM formulations were achieved by initial dissolution in cremophore (15%) and subsequent dispersion in water (85%). The animals were anesthetized during the PDT process using gas anesthesia (sevoflurane), while all other skin areas but the tumor with a 1–2 mm peripheral margin were appropriately shielded from light. All animal experiments were executed in compliance with institutional, national, and European guidelines and regulations and after approval from the institutional review board of Radium Hospital (application number: 7489).

    Reporting Summary

    Further information on research design is available in the Nature Research Reporting Summary linked to this article.


    20 Years of Photoshop | killexams.com real questions and Pass4sure dumps

    Digital Camera Home > Photoshop Turns TwentyPASINI REPORTS ps-logo.jpg

    By MIKE PASINIEditorThe Imaging Resource Digital Photography Newsletter

    Palace of Fine Arts party showcases 20 years of development -- and we're there with a Sony HX5V

    SAN FRANCISCO -- The fog could not obscure the significance of the twentieth anniversary of Adobe Photoshop's first release. As National Association of Photoshop Professionals President Scott Kelby observed, the software took image editing out of the hands of a small group of technicians running minicomputers and put it into the hands of artists.

    Photoshop 2.0 filters disk

    Twenty years later, no one's complaining.

    On Thursday, Feb. 18, roughly half the seats in the Palace of Fine Arts theater, which holds 979, were filled with Photoshop engineers, Adobe executives, photographers and painters who have built careers using a program that began life in a basement. Video of the event is available online.

    Originally the work of Thomas and John Knoll, the early version was no more than a file format converter named Display. John added gamma correction to properly display translated images on screen and there was no stopping after that.

    A LITTLE PERSPECTIVE | Back to Contents

    Our own association with Photoshop began after a seminar in 1991 in which Russell Brown showed off Version 2.0.1.b14 by demonstrating everything from using Levels to creating 3D images by shifting the red channel. He'd promised attendees a demo version but it didn't actually exist. Instead, he shipped the real thing to us.

    The digital camera of those days was a scanner. In fact the seminar was devoted to acquiring the right hardware, calibrating your display, scanning images and editing those scanned images for press.

    Notes from a long-ago seminar (Photoshop version number at top)

    The breakthrough Photoshop represented twenty years ago, however, wasn't as obvious then as it is today.

    At the time, we were helping a photo lab that was curious about moving retouching and image compositing out of the darkroom and onto computers. Photoshop wasn't in the running.

    But as we sat in on one presentation or another from firms trying to commercialize development begun for aerospace projects, we kept wondering if these very expensive systems couldn't be matched by a personal computer running Photoshop.

    Turns out they could.

    PAINTING FOR THE MASSES | Back to Contents

    But that professional use of Photoshop only foreshadowed adoption of the program by creatives of all stripes. The fun of drawing PostScript output with Illustrator for output to a PostScript printer had prepared graphic artists for the thrill of painting with Photoshop just a few years later.

    With both vector and bit-mapped imaging options available on an affordable platform, the creative community enjoyed a new suite of tools designed for the digital age. Before anyone thought to call it a "creative suite."

    And when digital photography came on the scene in the late 1990s, Photoshop was already there -- ready and waiting.

    The Palace of Fine Arts using Handheld Twilight mode

    THE PALACE | Back to Contents

    We arrived at the Palace of Fine Arts early but after the sun had set somewhere beyond the fog bank. The building itself, described by its creator Bernard Maybeck as beauty tempered by sadness, is illuminated by floodlights at night. So we wandered around taking a few shots of it, some using Sony's innovative Handheld Twilight mode, others just at high ISO.

    And we weren't alone. There were several other photographers aiming their glass at the colonnades and caryatids of the last remaining building from the 1915 Panama-Pacific Exposition, celebrating San Francisco's rebirth from the 1906 earthquake.

    OUR GEAR | Back to Contents

    Our glass happened to be a recently-arrived Sony HX5V review unit, a 10-Mp digicam with built-in HDR, Handheld Twilight mode and that Exmor-R sensor that can see in the dark with little noise at high ISO settings. All the shots in this article were taken with that camera.

    We shot everything in Program mode, with -1.75 EV because both the Palace and the theater were dark settings, relying on the multi-segment metering mode. We set ISO to 800 because we really didn't need ISO 1600.

    T-shirt toss for early (seat) adopters

    Although our shots for this article are crops resized to fit a 250-pixel maximum frame, the originals are remarkably clear and noise free. We'll be posting a few of them in full resolution in a gallery accompanying our review of the camera.

    The band kicks the evening off

    THE PARTY | Back to Contents

    The party kicked off with a video sketch of Kelby and crew trying to sell a Photoshop party to, well, Photoshop (played by a box). The sketch described all the ideas they had which gradually evolved into what was going to happen on stage.

    And that started with a musical number before Kelby took the stage, going through a typical image edit using Photoshop. The joke was that every tool he used to do the edit in Photoshop CS4 existed in the very first version.

    Even Gaussian Blur was in Version 1. And Levels, which Russell Brown has called the soul of Photoshop, was there, too.

    Jeff Schewe did a video tour of Version 1.0.7, released just five days after the originally shipping version. He had discovered it still runs on a G4 Mac running Tiger.

    Jeff Schewe

     

    Scott Kelby

    He pulled up "the most manipulated file in the history of Photoshop," known as Jennifer in paradise, a photo of John Knoll's future wife. He made a Levels adjustment, clicking the Preview button to see the effect of his changes. He cloned and painted clouds, loaded an alpha selection, flipped it and placed it by dropping the floating selection.

    FEATURES | Back to Contents

    Continuing the Photoshop story, Kelby promised (not) to go through each version in detail. Instead, he said he had asked various Photoshop mavens to demonstrate one feature from each release. Quickly. That turned out to be riotously entertaining, particularly if you lived through those days when graphic artists had to pull teeth without the benefit of anesthesia.

    Jennifer in paradise

  • Dave Cross began the tour by showing how to do a drop shadow in Version 2 with no Layers and no Editable Type.
  • Matt Kloskowski demonstrated Layers in Version 3, bringing back detail from a background layer using the Blend If option in the Layer Style palette.
  • Adobe's Julieanne Kost showed off Version 4's Actions, Guides, the Navigator Panel, Adjustment Layers and Gallery Effects, among other new features like Free Transform and Gradients.
  • Rafael "RC" Concepcion worked with Version 5's History Brush. This version also introduced Editable Text, Color Management and the History Brush.
  • Adobe's Terry White discussed a few ways to use Version 5.5's Extract filter (and where to find it in CS4). And the Background Eraser's benefits.
  • Corey Barker demonstrated Version 6's Layer Styles by comparing how to modify type before Layer Styles and after.
  • Kloskowski returned to show off the Healing Brush in Version 7, building a torn edge on an image.
  • Kelby showed Adobe Camera Raw's retouching prowess in the first version of the Creative Suite, turning a bland male portrait into a pseudo-HDR shot and then laboring over every blemish on a female face.
  • Cross returned to show CS2's Smart Objects, setting up a multi-image layout and, by opening the image as a Smart Object being able to reuse the layout.
  • Barker demonstrated CS3's 3D editing capabilities on a logo.
  • And Concepcion concluded the feature fest with a demo of CS4's content-aware scaling, fitting his 4:3 photos onto paper sizes of other aspect ratios.
  • Dave Cross

     

    Matt Kloskowski

    Julieanne Kost

     

    RC Concepcion

    Terry White

     

    Corey Barker

    RUSSELL BROWN | Back to Contents

    But wait, there's more, as Russell Brown famously likes to say. And Russell Brown himself was the "more" this time.

    After an old video clip of a younger Brown demonstrating how to paste himself into a photo of Ron and Nancy Reagan, Brown himself took the stage not at the podium to command some laptop but at a desk where his magical Epson overhead projector had been set up.

    Brown sits down at his overhead projector

    One of the first things he examined was his iPhone, which had Photoshop 1.0.7 on it. He too brought up Jennifer in paradise and adjusted Levels. But that's about all this app imitation could do. It was built in just a few days by Ansca using its Corona iPhone development platform. And 40 people in the audience won a copy on a 2-GB USB thumbdrive attached to their chair. Including the guy sitting next to us.

    Then Brown went back to the dawn of man, the beginning of time, way back when "they used real mice to start their presentation" (as he displays a toy mouse in his hairy ape mitts on the projector). The presentation? Oh, the history of Photoshop.

    3D into 2D without glasses

    He displayed stone interfaces for Photoshop 2.0 during "early slate computing" in which the cave painters had Macs and the stone counters used PCs.

    Layers in Photoshop 3 were demonstrated with a layered Photoshop startup screen. Version 4 showed Layers by magically turning an image of dominoes into real ones that fall down and recompose themselves into an image.

    Brown slid a slice of bread on the projector's table and used French's mustard to write "PS 5" on it ("in Frankfurter typeface"). Then he picked it up and took a bite out of it before returning the partly-consumed slice to the table. "Edible text," he mumbled, illustrating the editable text feature introduced in Photoshop 5. Then he used Undo to restore the bite.

    Brown prepared for the future

    But wait, he bit into it three more times and undid it all, too. "Multiple Undo!" he explained.

    Version 6 used an overlay of an animated blender to manage color. Nobody remembered that feature, though.

    The Healing Brush restored his amputated hand in Version 7, even cleaning up the green blood.

    The first Creative Suite version used his glasses to show Lens Blur and how to make money with Photoshop by magically upgrading coins until he got to paper money, which CS wouldn't reproduce.

    CS 2 showed off Noise Reduction with a marker interface that took you from street level to outer space, where it's much quieter.

    The Auto Blending demo for CS 3 merged slices of an image into a photo of an auto that turned into a 3D model that drove off the table.

    Content-aware scaling in CS 4 was illustrated by an overlay of a bathroom scale that reported not the weight of what had been put on it but the name of the object. When Brown put his watch on it, for example, it dialed in "Russell's Watch." When he put a picture of Steve Jobs on it, it swung wildly to the right over a word we can't recall before settling on "Steve Jobs."

    And in the future? A future X-Acto blade cuts out the globe in a shot from outer space and automatically fills it in with space, a black hole, that sucks us all in.

    THE FUTURE | Back to Contents

    Senior Vice President and General Manager of Adobe's Creative Solution's Business Unit John Loiacono came to Brown's aid just before he was sucked into his own black hole. "We're done with your analog world," he told Brown, sending him off.

    Instead, Loiacono said, we need a digital equivalent to fill Brown's black hole. He introduced Kevin Connor, Adobe product manager for all things digital, to demonstrate how fills can be context aware.

    Loiacono with Connor

    Connor introduced the audience to PatchMatch technology, which treats your image like a set of puzzle pieces. You can remove a selection from the scene without leaving a hole or move a tower to a different part of the building, shuffling the building parts around.

    "Let's ship it," Loiacono said. But Connor demurred, suggesting it needed to be polished a bit. This led to a short private chat (overheard by all) in which Connor was convinced to give the people something now. Right now.

    So Connor opened an image of a fellow standing in front of a stone wall. He selected the figure and expanded the selection a few more pixels before removing the guy from in front of the wall. Instead of leaving a white hole in the image, PatchMatch seamlessly filled the cut with stones. Applause.

    Thomas and John Knoll (before the HX5V knew where they were)

    "But wait! There's more," Loiacono prompted.

    Connor then showed a panorama that had been stitched together with a very uneven border. You can crop it, of course, but using PatchMatch, the gaps around the edges were credibly filled. Applause.

    "OK, Kevin, you can keep your job another week," Loiacono promised.

    Loiacono then introduced the Knoll brothers who had started all this. Yes, applause. They modestly stood up, turned around and sat down faster than the HX5V could focus on them, unfortunately, but the blurred image has a sort of ghostly poetry to it.

    A few of the engineers

    And then he asked the nearly 70 engineers working on Photoshop now to stand up and take a bow. And yes, more applause.

    Loiacono went through each version of Photoshop's code names and then thanked Kelby for his organization's work in supporting the product. Kelby told everyone there was a T-shirt and poster for them in the lobby and brought out the band for one last tune before everyone filed for the exits.

    The poster

    THE COOL NIGHT | Back to Contents

    The fog had lifted by the time we left the theater. Entertaining and amusing as the party had been, we were surprised to find ourselves feeling a little sad.

    Revisiting the glow of Photoshop 2.0, 2.5, 3.0 reminded us of those days and nights at the nine-inch black and white monitor of a Macintosh SE. Saving to floppy disks. Before some long-ago deadline. For clients no longer in business.

    We felt a little like those beautiful women who look over the ramparts of the Palace of Fine Arts, weeping. Beauty tempered by sadness.

    But, we thought as we walked on, we are still launching Photoshop and buffing up our images to a high gloss, creating in our small bunker work that makes us smile.

    So make a wish and blow out the candles, Photoshop. We'll take the picture.



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