Fig. 5. S6K1/2 regulates the SASP without affecting the growth arrest in human fibroblasts undergoing OIS.
a, Experimental scheme. IMR90 fibroblasts were stably transduced with the pLNC-ER:RAS retroviral vector and treated with 4OHT for senescence induction. b, Representative IF staining of BrdU, p16INK4A, IL-1α, IL-1β and IL-8 after 7 d (BrdU and p16Ink4a) or 8 d (SASP) with or without 4OHT treatment in IMR90 ER:RAS cells. Scale bar, 100 μm. c–e, IMR-90 ER:RAS cells were reverse transfected with either AllStars (scrambled sequence, siControl) or the indicated siRNAs. Cells were treated with or without 4OHT on the following day to induce senescence. Quantification of IF staining for BrdU incorporation (c), p16INK4A (d) and p21CIP1 (e) after 5 d of 4OHT treatment (n = 5 biological replicates from two independent experiments). f–i, Relative mRNA expression for pro-inflammatory SASP components (IL1A, IL1B, IL8, CCL20) assessed by RT–qPCR after 4 d of 4OHT treatment with the indicated siRNAs (siControl, siS6K1_2, siS6K1_4, siS6K2_4 and siS6K2_5 n = 4 for IL1A, IL1B and IL8 and n = 3 for CCL20; siS6K1/2 and siC/EBPβ n = 3 for IL1A, IL1B, IL8 and CCL20) in IMR90 ER:RAS cells. mRNA expression was normalized to the Rps14 housekeeping gene. n denotes independent experiments. Data are expressed as mean ± s.e.m. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparison test (c–i). j, Immunoblot images of a single experiment of S6K1, S6K2, IL-1β, IL-8 and GAPDH after 7 d of 4OHT treatment with the indicated siRNAs in IMR90 ER:RAS cells. IL-8 and GAPDH (loading control) were run on the same blot. S6K1, S6K2 and IL-1β were run on separate blots; therefore, GAPDH served as a sample preparation control for those blots.