Supplementary Figure 5: ZFP36L1 regulates the SASP.
From: mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype
(a) Serine54 in ZFP36L1 was identified as an mTOR inhibition-sensitive phosphosite by phosphoproteomics (n = 3). (b) Extension of Fig. 5b. Specificity of MK2 inhibitor III is proved by the decrease in the phosphorylation of HSP27 (pHSP27S82) a downstream target of MK2. (c) Schematic model of ZFP36L1 activity and protein stability. (d) Percentage of ZFP36L1 mRNA in translating polysomes during OIS (n = 3). (e) IMR90 ER:RAS cells were infected as indicated. Immunostainings were performed 7 days after 4OHT induction. Fold changes of normalised intensity values are shown (n = 4). (f) SASP genes expression was measured in IMR90 fibroblasts 8 days after irradiation (5 Gy) (n = 3). (g) Designed TTP and ZFP36L2 phosphomutants. As MK2 phosphosites for TTP and ZFP36L2 have not been described, we aligned their sequences with that of ZFP36L1 aiming to identify putative MK2 phosphosites. In the case of TTP, homology is low. However, the two known MK2 phosphosites in the murine form of TTP (ref. 59) are conserved in the human form. (h,i) IMR90 ER:RAS cells were infected with ZFP36L1wt/mut, ZFP36L2wt/mut or TTPwt/mut. OIS was induced by 4OHT for 7 days. (h) Protein levels were analysed by immunoblot in presence of MG132 (2 μM, 12 h). (i) Expression of SASP genes was measured by qRT-PCR (n = 3). (j) SASP components detected in our proteomics analysis that are downregulated by mTORinhibitors, shmTOR and ZFP36L1Mut. (k) Venn diagrams representing the overlap between SASP factors upregulated by mTOR inhibitors, shmTOR and ZFP36L1Mut. (l) ARE Score for SASP factors: (left) commonly upregulated by shmTOR and mTOR inhibitors; (right) commonly downregulated by shmTOR, mTOR inhibitors and ZFP36L1Mut. Plots denote ARE score distribution for 105 random combinations of 15 and 17 mRNAs respectively. Statistical significance was calculated by using: (d,e,j and i): Student’s t-test (k: hypergeometric test), (l: permutation tests). ***P < 0.001; **P < 0.01; *P < 0.05; NS, non significant. Error bars represent means ± s.d. For b and h data are representative of 3 independent experiments. n represents number of independent experiments. For unprocessed original scans of blots, see Supplementary Fig. 9. For raw data, see Supplementary Table 7.
