Supplementary Figure 3: Depletion of nucleolar proteins leads to senescence in IMR90 cells. | Nature Cell Biology

Supplementary Figure 3: Depletion of nucleolar proteins leads to senescence in IMR90 cells.

From: Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway

Supplementary Figure 3

(a) Proteomic analysis of RAS-senescent IMR90 cells treated with MG132 (10 μM, 18 hours) or vehicle. 1018 proteins were enriched in MG132-treated cells compared to control. Proteins associated to protein synthesis machinery and the indicated subcategories were retrieved using the Babelomics 4.3 platform. (b-d) QPCR for the indicated genes performed with total RNA extracted from (b-c) cells at day 20 post-infection expressing (b) H-RASV12 (RAS), (c) PML-IV (PML) or an empty control vector (Vect) and from (d) uninfected young (passage 24) and old (passage 38) IMR90 cells. Data were normalized over TBP and HMBS and presented as means relative to vector-infected cells or young cells, (n = 1). (e-g) QPCR for the indicated genes performed with total RNA extracted from cells expressing shNTC (non-targeting shRNA); shR-A or -B (shRNAs against RSL1D1); shN-A or -B, (shRNAs against nucleostemin); shD-A or -B, (shRNAs against DDX21); shE-A or -B, (shRNAs against EBP2) at day 14 post-infection. Data were normalized over TBP and HMBS and presented as mean relative to shNTC infected cells, (1 out of 2 independent experiments with similar results). (h) SA-β-gal of cells expressing the indicated shRNA expression vectors and fixed at day 14 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). (i-j) QPCR for p21 and p16INK4a performed with total RNA extracted from cells as in (e-g). Data were normalized over TBP and HMBS and presented as means relative to shNTC infected cells, (1 out of 2 independent experiments with similar results). (k) Indirect immunofluorescence with specific anti-fibrillarin (FBL) and anti-RPL29 antibodies followed by confocal microscopy of cells as in (h) and fixed at day 7 post-infection. Data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of positives cells for nucleolar localization of RPL29, (1 out of 3 independent experiments with similar results).

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