Extended Data Fig. 2: Treatment with 3DA alleviates oncogene-induced senescence.
a, Representative immunofluorescence images of p16INK4a (red) in IMR90 ER:RAS cells cells 4 days after treatment with 4OHT and 10 μM 3DA or vehicle (DMSO). Scale bar, 100 μm. b, Quantification (n = 3). The statistical significance was calculated using unpaired two-tailed t-test. c, p21CIP1 protein expression in IMR90 ER:RAS cells treated with DMSO or 4OHT to induce senescence or 4OHT and 10 μM 3DA. Normalized nuclear intensity values and mean values on day 3 (left), day 6 (middle) and day 9 (right panel) are shown (n = 200 cells per condition). The statistical significance was calculated using unpaired two-tailed t-tests. d, Timeline of the experiment (left) and crystal violet staining (right). IMR90 ER:RAS cells were treated with 4OHT continuously to induce senescence. On day 6, DMSO or 10 μM 3DA were added (change of media every 3 days). Cells were fixed on day 14. e, Timeline of the experiment (left) and quantification of immunofluorescence staining for p16INK4a (middle) and BrdU (right). IMR90 ER:RAS cells were treated with DMSO for 4 weeks, or 10 μM 3DA for 2 weeks followed by DMSO for 2 weeks, or 10 μM 3DA for 4 weeks. Media was changed every three days. Treatment was started when cells were at passage 14 and ended when cells were at passage 20. At the end of the experiment, p16INK4a protein expression and BrdU-positive cells were quantified. For p16INK4a, normalized nuclear single-cell intensity values and mean of 200 cells are shown. To assess proliferation, percentage of BrdU-positive cells was measured (n = 3). Statistical significances were calculated using one-way ANOVA. All error bars represent mean ± s.d; n represents independent experiments unless otherwise stated.
