Supplementary Figure 6: Further characterization of the role of ZFP36L1 in the regulation of the SASP.

(a) GSEAs of a signature associated with senescence⁶⁰ and (left) SASP components upregulated in OIS (ref. 6; right) in the gene expression profile (GEP) of IMR90 ER:RAS cells expressing ZFP36L1^Mut versus control IMR90 ER:RAS, both 4OHT-treated. NES, normalized enrichment score; FDR, false discovery rate. GEPs were obtained by performing transcriptome sequencing (RNA-Seq) of the aforementioned conditions in n = 3 independent experiments. RNAs were collected 7 days after induction with 4OHT. (b) Mean ARE score of genes down regulated (at the indicated log₂ of fold changes) when comparing IMR90 ER:RAS cells expressing ZFP36L1^Mut versus control IMR90 ER:RAS, are shown in blue. The distribution of mean ARE scores derived from 10⁵ simulations of random genes with equal sample size are shown in red. (c) This is an extension of Fig. 6c. The expression of TIMP was monitored by qRT-PCR at the indicated times. Data are for a representative experiment of n = 2 independent experiments. (d) Brdu incorporation was measured by IF at the indicated times. Error bars represent the ±s.d. from n = 3 independent experiments. Student’s t-test was performed to compare ZFP36L1^Mut expressing cells (200 ng ml⁻¹ doxycycline) to the control senescent cells (vector, +4OHT). ***P < 0.001. (e,f) IMR90 ER:RAS expressing TRE–FLAG-ZFP36L1^Mut (or empty vector) and the reverse transactivator (rtTA-M3) were further infected with a vector encoding the coding sequence of p21. After 6 days of incubation with 4OHT, the expression of ZFP36L1^Mut was induced with 200 ng ml⁻¹ doxycycline for 72 h and (e) the % of BrdU positive cells or (f) SA-β-Gal positive cells was quantified. Error bars represent the ±s.d. from n = 4 independent experiments. SA-β-Gal stainings are representative of n = 3 independent experiments. Student’s t-test was performed to compare the indicated conditions ***P < 0.001; NS, non significant. For raw data, see Supplementary Table 7.