Supplementary Figure 5: CDK4 and CDK6 bypass RSL1D1 knockdown-induced senescence.

(a) SA-β-gal of IMR90 cells expressing the indicated shRNA expression vectors in combination with the expression of CDK4 (1 out of 3 independent experiments with similar results), CDK6 (n = 1) or a control vector (Vect) at day 20 post-infection. Data were quantified from >5-independent cell counts up to a total of at least 150 cells and are presented as the mean percentage of positive cells. shNTC: non-targeting shRNA; shR-A or -B: shRNAs against RSL1D1. (b) Growth curves of IMR90 cells as in (a). Data are presented as means normalized to day 0 of each condition. (1 out of 3 independent experiments with similar results for CDK4) and (1 out of 2 independent experiments with similar results for CDK6). (c-h) QPCR for the indicated genes performed on reverse transcribed total RNA extracted from cells as in (a) at day 20 post-infection. Data were normalized over TBP and HMBS and presented as means relative to vector/shNTC infected cells, (1 out of 2 independent experiments with similar results). (i) SA-β-gal of IMR90 cells expressing the indicated shRNA expression vectors in combination with the expression of 3xFLAG-CDK4(WT), 3xFLAG-CDK4(K35M) or an empty control vector (Vect) at day 20 post-infection. Data were quantified from >5-independent cell counts up to a total of at least 150 cells and are presented as the mean percentage of positive cells, (1 out of 2 independent experiments with similar results). shNTC: non-targeting shRNA; shR-A or -B: shRNAs against RSL1D1. (j-m) QPCR for the indicated genes performed on reverse transcribed total RNA extracted from cells as in (i). Data were normalized over TBP and HMBS and presented as mean relative to vector/shNTC infected cells, (1 out of 2 independent experiments with similar results).