Research Paper Volume 11, Issue 2 pp 549—572

S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α

Figure 2. S100A13 modulates NF-κB activity and SASP expression during cell senescence. (A) HCT116 cells were transfected with the control vector, S100A13 wild type or mutant type, and treated with Dox (100 nM) for 3 days. Then cell lysates were subjected to western blot analysis using the indicated antibodies. (B) ER:Ras IMR90 cells stably transduced with PHBLV, S100A13 wild type or mutant type were given 4-OHT for 6 days. Cell lysates were then subjected to Western blot analysis for the indicated proteins. (C) HCT116 cells were transfected with control (siNC) or two independent siRNAs against S100A13, and treated with Dox (100 nM) for 4 days. Then cell lysates were subjected to western blot analysis using the indicated antibodies. (D) ER:Ras IMR90 cells stably transduced with control (siNC) or two independent shRNAs against S100A13, were given 4-OHT for 6 days. Cell lysates were then subjected to Western blot analysis for the indicated proteins. (E and F) HCT116 cells were transfected with vector, S100A13 wild type or mutant type, or transfected with control (siNC) or two independent siRNAs against S100A13, and treated with Dox (100 nM) for 4 days. Then mRNA levels of some SASP genes were analyzed by real-time qPCR (n=3). (G) HCT116 cells were transfected with wild type S100A13, and treated with Dox (100 nM) for 3 days. Control IgG (0.6 ug/ml) or neutralizing antibody IL-1a (0.6 ug/ml) were added for the last 2 days. Then cell surface-bound IL-1α were analyzed by FACS (n=3). (H) HCT116 cells were transfected with siRNA#2 against S100A13, and treated with Dox (100 nM) for 3 days. Solvent or recombinant IL-1a protein (300 ng/ml) were added for the last 2 days. Then cell surface-bound IL-1α were analyzed by FACS (n=3). Three independent experiments were performed. Error bars represent means ± SD (n = 3) *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 in (E–H).