Figure 4. Prostaglandins promote cellular senescence phenotypes.
A-F. IMR-90 fibroblasts were induced to senesce by IR and treated with DMSO or COX-2 inhibitors CAY-10404 (CAY) or NS-398 (NS) for 10 days. A. Conditioned media were harvested and secreted PGD2 was measured by ELISA. B. 24 h EdU incorporation for irradiated cells in each treatment group. C. SA-Bgal positivity for irradiated cells in each treatment group. D. p21 mRNA levels in each treatment group. E. p16 mRNA levels from each treatment group. F. Conditioned media from (A) were analyzed for IL-6 secretion by ELISA. G. mRNA levels of genes encoding SASP factors from all treatment groups were measured by qPCR. H-N. Proliferating cells were cultured for 10 days in the presence of DMSO or 5 μM 15d-PGJ2 or dihomo-15d-PGJ2. H. 24 h EdU labeling indices for each treatment. I. Example of SA-Bgal positive cells after each treatment. Panels are cropped zooms to larger images, and red saturation has been lowered for all images in order to allow visualization of the blue x-gal staining. J. SA-Bgal positivity for each treatment. K. p21 mRNA levels in extracts from prostaglandin- and DMSO-treated cells. L. p16INK4a mRNA levels in extracts from prostaglandin-and DMSO-treated cells. M. LMNB1 mRNA levels in extracts from prostaglandin- and DMSO-treated cells. N. SASP factor PTGS2, PTGDS and PTGES mRNA levels were measured by qPCR. Normalized to actin. O. Cells were transduced with lentiviruses expressing shRNA targeting either GFP (shGFP; control) or PTGDS (shPTGDS), irradiated as in (A), and mRNA extracted 10 d after irradiation. SASP gene expression was measured by qPCR. Bar graphs indicate averages of at least 3 experiments (* = p<0.05, ** = p<0.01, *** = p<0.001, unpaired, 2-tailed t test). Heat maps indicate averages of 3 experiments (* = p<0.05, 2-way ANOVA). See also Figure S4.