Fig 2: SASP CM causes cell death by pyroptosis.A, MCF-10A cells were treated with CTRL or SASP CM collected from BIS or OIS fibroblasts with or without MCC950 (10 µM), an NLRP3 inhibitor. Cells were imaged using immunofluorescence microscopy. Images were taken at 10× and the scale bar is 100 µm. Cells were stained with Hoechst and SYTOX Green. Quantification of the percentage of SYTOX Green positive cells out of the total number of cells was depicted. B, MCF-10A cells were engineered using shRNA against caspase-1 and immunoblotted to validate the reduction in caspase-1 protein. C, EV control or caspase-1 shRNA-transduced cells were treated with CTRL or SASP CM from BIS or OIS and imaged using immunofluorescence microscopy. Cells were stained with Hoechst and SYTOX Green. Quantification of the percentage of SYTOX Green positive cells out of the total number of cells was depicted. D, MCF-10A cells were engineered using shRNA against GSDMD and immunoblotted to validate the reduction in GSDMD protein. E, EV control or GSDMD shRNA–transduced cells were treated with CTRL or SASP CM from BIS or OIS and imaged using immunofluorescence microscopy. Cells were stained with Hoechst and SYTOX Green. Quantification of the percentage of SYTOX Green positive cells out of the total number of cells was depicted. Sytox data were analyzed by two-way ANOVA followed by Tukey comparison test, where ns is no statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are presented as mean ± STDEV. Graphs are representative data collected from a minimum of three biological replicates. F, MCF-10A cell lysates were treated with CTRL or SASP CM from BIS fibroblasts and immunoblotted for full-length (FL) and cleaved (c) GSDMD. Densitometry is represented as fold change relative to EV control. BIS, bleomycin-induced senescence; CM, conditioned media; EV, empty vector; GSDMD, gasdermin D; OIS, oncogene-induced senescence; SASP, senescence-associated secretory phenotype.

Fig 3: (A) Western blot analysis images and correlation histograms of Caspase-1, GSDMD-N proteins in osteoblasts of mouse BMSCs cells after 7 days of osteogenic differentiation induction. (B) BMSCs cells after induction of osteogenic differentiation were subjected to qRT-PCR to compare the relative expression levels of Caspase-1, GSDMD-N mRNA in each group. (C) LDH activity was measured in the supernatant of osteoblasts. (D) Fluorescence microscopy images of osteoblasts subjected to Hoechst 33342/PI double staining, scale bar = 200 µm. All experiments were performed independently at least three times. (****P<0.0001 vs Control group; ###P<0.001, ####P<0.0001 vs OVX group).