Lsm510 confocal laser microscope

Cells were seeded at 10⁴ cell density on coverslips in 6-well plates. Cells were treated with MMC, fixed with 3.7% formaldehyde for 10 min in the dark at room temperature and permeabilized with 0.3% Triton-X for 30 min at 37°C. Cells were incubated with γH2AX antibody (Millipore) for 1 h at 37°C followed by Alexa Fluor 488 F anti-mouse secondary antibody for 1 h in the dark at room temperature. The nuclei were stained with DAPI (Invitrogen) and mounted for analysis with a Zeiss LSM-510 Confocal Laser Microscope. Approximately 100–150 cells were used for counting the number of foci using ImageJ 1.47a software (NIH, Bethesda, MD; http://imagej.nih.gov/ij/).

MIA PaCa-2 cells were cultured at 5,000 cells per 8mm coverslip. After treatment, cells were fixed, permeabilized, stained and mounted as previously described (Primary- γH2AX; Millipore; 1:500, HuR; 1:200; Santa Cruz Biotechnology; Secondary- Alexa Fluor 488 F anti-mouse; DAPI ProLong Gold, Life Technologies). Coverslips were imaged with a Zeiss LSM-510 Confocal Laser Microscope and Image J was used for foci counting, as previously reported (17 (link), 20 ).

Images were acquired with a Zeiss LSM510 confocal laser microscope. On the 12 mm diameter coverslip, 5 horizontal sections were observed and myotubes were counted. Three replicates were done of each staining experiment and error bars were calculated using standard deviation from the mean. ImageJ software was used to quantify KDM4E RNAScope and LEUTX antibody staining signals in LEUTX and DUXA depletion experiments. Briefly, line was drawn around each myotube, and the fluorescent signal was measured inside of the line as the mean gray value and the average background mean gray value from three different negative myotubes was subtracted. Because so few myotubes were positive for KDM4E RNA and LEUTX protein to begin with, and to be more comparable with RT-qPCR results, the integrated density (the total KDM4E RNA or LEUTX protein signal in each myotube) was then back-calculated as the mean intensity multiplied by the area of each myotube. The integrated density values of top 5% myotubes (corresponding to 25 myotubes out of 500 myotubes examined) amount to >95 % of the total KDM4E RNA or LEUTX protein signals in control shRNA-treated myotubes. Thus, top 5% values in each group were used for graph and data analysis. Arbitrary numbers in Y-axis was obtained by normalizing all the data to the mean value of the control shRNA samples.

Approximately 50,000 cells were plated on coverslips per well in 24-well plate. After 24 h, cells were treated with MMC, gemcitabine and oxaliplatin for 24 h. Cells were then washed 2 times with 1X PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.25% Triton-X for 30 minutes, blocked with 5% goat serum for 1 h, and incubated overnight with HuR antibody, as previously described (14 (link)). DAPI was used to stain cell nuclei and coverslips were mounted with DAPI ProLong Gold Antifade (Invitrogen). Slides were visualized with a Zeiss LSM-510 Confocal Laser Microscope. All images were taken at 40X magnification with oil. Images were cropped using AIM browser.