Supplementary Figure 8: mTOR inhibition blunts the tumour suppressive effects of the SASP.
From: mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype
(a) CMs of IMR90 ER:RAS expressing TRE– ZFP36L1Mut (or empty vector) and p21 (or empty vector) were collected 72 h after induction of ZFP36L1Mut and 9 days after 4OHT addition. The effect CMs had on BrdU incorporation and SASP induction in IMR90 wt cells was evaluated by IF. Graphs show data from n = 4 independent experiments. (b) IMR90 wt cells were co-cultured with the indicated IMR90 ER:RAS cells with or without 4OHT. BrdU incorporation was monitored by IF. Data are representative of n = 2 independent experiments. (c) This figure is related to Fig. 8c. Expression of the indicated transcripts was quantified by qRT-PCR. Carrier, n = 5 mice; Rapamycin, n = 4 mice. P-values are included. (d) Expression of MAPKAPK2, 4EBP1 and phosphorylation of 4EBP1 were analysed by immunoblot. Quantification is presented in Fig. 8d. (e) NrasG12V transposons were co-delivered into mouse livers through hydrodynamic injection (Day 0). Mice were sacrificed 3 days later and Nras+ cells were counted (n = 4 mice per condition) Mice were treated with carrier or drugs from 3 days prior NrasG12V injection. Representative images of the sections quantified are shown (200x). Scale bar, 100 μm. (f) Quantification of the expression of immune cell markers by qRT-PCR in livers of the indicated mice 6 days after NrasG12V injection. Carrier, n = 5 mice; rapamycin, n = 4 mice. CD3, T cell marker; B220, B cell marker; CD68, macrophage marker; KlrD, NK cell marker. P values are included. (g) NrasG12V transposons were co-delivered into mice as explained before. Mice were sacrificed 12 days later. Mice were only treated with carrier or rapamycin 3 days post-injection Nras+ (left), p21Cip1+ (centre) and p16Ink4a+ (right) cells on liver sections were counted (n = 4 mice per condition). Representative images of sections quantified in the top panel are shown in the bottom. Scale bar, 100 μm. Statistical significance was calculated using Student’s t-test, ***P < 0.001; **P < 0.01; *P < 0.05; NS, non significant. Error bars represent means ± s.d. For unprocessed original scans of blots, see Supplementary Fig. 9. For raw data, see Supplementary Table 7.
