Fig. 1

ATRX is necessary for doxorubicin-induced senescence. LS8817 cells were transduced with either a scrambled (shSCR) or ATRX-specific (shATRX) lentiviral knockdown vector and subsequently treated with 100 nM doxorubicin for 7 days. a Extracts were made from the cells indicated above each lane and the expression of proteins determined by immunoblot as indicated on the left of each panel. ATRX was detected with the D-5 antibody. b Cells were treated as described on the left and stained with the antibodies indicated on top of each panel. Representative images are shown (top) and quantification of the foci is plotted (bottom). c The indicated cells were plated on day 0 and either treated with 100 nM doxorubicin (doxo) or left untreated (CTRL). Cell number was counted at the indicated days and the relative number of cells is plotted. d, e The accumulation of SA-β-gal-positive cells (d) or SAHF-positive cells (e) for each individual treatment condition was measured. Representative SAHF images are shown (below). f The accumulation of four of the liposarcoma SASP transcripts was measured by qPCR under each indicated condition and their induction in doxorubicin treated cells relative to that in untreated cells is plotted and compared. g The cells were treated with 100 nM doxorubicin (7D doxo) or left untreated (CTRL) for 7 days. Cells were then collected, counted and replated at low density in the absence of drug. Clonogenic growth was measured by crystal violet staining 3 weeks later. Representative images from two independent experiments are shown. Colony number is not quantified as individual colonies could not be discerned. All data are plotted as mean ± SEM from at least three independent experiments. Asterisk indicates p < 0.05 using a two-sided Student’s t-test. All images were taken at the same magnification. The scale bar is 20 microns. See also Supplementary Fig. 10 for uncropped blots