Supplementary Figure 6: RSL1D1 knockdown-induced senescence is not affected by CDK4 inhibitors p16INK4a and p21.

(a-b) Growth curves of IMR90 cells expressing combinations of the indicated shRNAs. Data are presented as means normalized to day 0 of each condition. (1 out of 2 independent experiments with similar results). shNTC: non-targeting shRNA; shp16: shRNA against p16INK4a (CDKN2A); shp21: shRNA against p21 (CDKN1A); shR-A or -B: shRNAs against RSL1D1. (c-e) QPCR for the indicated genes performed on total RNA extracted from cells, as in (a-b), at day 20 post-infection. Data were normalized over TBP and HMBS and presented as means relative to shNTC/shNTC infected cells, (1 out of 2 independent experiments with similar results). (f) Immunoprecipitation with pre-immune serum or with RPS14 antibody in H1299 cells. Lysates and immunoprecipitates were immunoblotted for the indicated proteins (1 out of 3 independent experiments with similar results). (g) SA-β-gal of IMR90 cells expressing RPS14-Myc or an empty control vector (Vect) at day 12 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 3 independent experiments with similar results). (h) Immunoblots for the indicated proteins for cells as in (g) at day 7 post-infection, (1 out of 3 independent experiments with similar results). Western blot panels for Myc and tubulin are the same as in Fig. 6b. (i) QPCR for the indicated genes performed on total RNA extracted from cells as in (g) and at day 9 post-infection. Data are normalized over TBP and HMBS and presented as mean relative to vector infected cells, (1 out of 2 independent experiments with similar results). (j-k) Indirect immunofluorescence (IF) with specific anti-53BP1, anti-γH2A.X, anti-RPL29 and anti-PML antibodies as indicated and nuclear counterstaining with DAPI. Images represent IMR90 cells expressing RPS14-Myc, p16INK4a or an empty control vector at day 14 post-infection, (1 out of 3 independent experiments with similar results). (k) IF data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of cells with positive nucleolar localization of RPL29. (l-n) IF foci data in cells as in (j-k) were quantified from 100 cell counts in triplicate and are presented as the mean. Quantification of the percentage of cells with 0-1, 2-5 or more than 5 foci of (l) 53BP1 and (m) γH2A.X per cell. (n) Quantification of the percentage of cells with 0-5, 6-10 or more than 10 foci of PML per cell. Unprocessed blots can be found in Supplementary Figure 9.