Fig 2: Activated caspases-3 and -7 contribute to pyroptosis in macrophage/microglia in multiple sclerosis. MS neuropathogenesis is driven by converging disease processes that create a proinflammatory microenvironment, including infiltration of peripheral immune cells, demyelination (1), axonal loss and neuronal cell death (2), and microglial activation (3). Widespread cell death releases proinflammatory DAMPs and alarmins (e.g., extracellular ATP), which trigger pyroptosis in macrophages (Mφ)/microglia (MG). Pyroptosis is lytic and highly proinflammatory, creating a positive feedback loop to drive further pyroptotic cell death. Within the pyroptotic cell (4), inflammasome activation leads to the cleavage and activation of proinflammatory cytokines (IL-1β and IL-18) and the cytotoxic pore-forming protein GSDMD, and as shown in this manuscript, procaspase-3. While activated GSDMD translocates to the plasma membrane to form pores, cleaved caspase-3 traffics to multiple cellular compartments wherein it cleaves several substrates that promote pyroptosis. In the nucleus, caspase-3 and -7 substrates DFF45 and PARP are cleaved and activated. ROCK1 is cleaved and activated in the cytoplasm and appears to concentrate around the cell periphery and in pyroptotic bodies, suggesting a putative role in pyroptotic body formation that mimics its role in apoptotic body formation during apoptosis. Collectively, the proteolytic network engaged by caspase-3/7 during pyroptosis activates parallel processes that facilitate cellular demolition, pyroptotic body formation, and membrane lysis during pyroptosis

Fig 3: BCL-2 family members are elevated in senescent cells and provide resistance to apoptosis.(a) IMR-90 human fibroblasts that were induced to senesce either through DNA damage (DIS), replicative exhaustion (RS) or oncogene expression (OIS), as well as controls proliferating cells (growing, G) and empty vector-transfected (V) cells, were treated for 10 h with TNF-a and CHX (TNF-a) or with vehicle (DMSO). Cell survival relative to vehicle-treated cells was determined by quantification of the remaining adherent cells. Histograms indicate the percentages of surviving senescent (DIS, RS and OIS) cells compared with G or V controls. Data are presented as mean±s.e.m of three repeats, performed in triplicates. (b) Western blot analysis of cleaved PARP, ICAD and caspase-3 proteins from senescent and control IMR-90 cells (labelled as in a), indicating levels of apoptosis after treatment with TNF-a and CHX. (c) Percentage survival of senescent and control cells (labelled as in a) 24 h after UV irradiation (80 J m-2). Data are presented as mean±s.e.m of three repeats, performed in triplicates. (d) Expression of BCL-2 family members (BCL-W, BCL-XL, BCL-2 and MCL-1) and of senescence effector proteins (p16, p21, p53) in senescent (DIS, RS, and OIS) and control (G, V) cells of both human (IMR-90) and mouse (MEF) origin. (e) Percentage survival of senescent and control cells (as in a) as well as in quiescent (Q) control cells after treatment for 24 h with the indicated concentrations of ABT-737, an inhibitor of BCL-W, BCL-XL and BCL-2. Data are presented as mean±s.e.m of three repeats, performed in triplicates. (f) Percentage survival of senescent (DIS and OIS) and control (G and V) cells treated with ABT-737 (10 µM) for 24 h with or without pretreatment for 6 h with the pan-caspase inhibitor z-VAD-fmk. Data are presented as mean±s.e.m of three repeats, performed in triplicates. (g) Western blot analysis of cleaved PARP and caspase-3 in the samples described in f. Data in b,d,g are representative blots of at least two independent experiments each. Data were analysed using Student's t-test. *P<0.05, **P<0.005.

Fig 4: miR-210 attenuates the hypoxia-induced increase in DNA fragmentation through the inhibition of GSK3ß kinase activity. (A) Quantitative TUNEL assay determining apoptotic DNA fragmentation, expressed as experimental blank-corrected absorbances (O.D) measured at ?450 (450 nm). (B) Qualitative Western blot depicting the processing of the DFF45 into cleaved-DFF45 (p11 fragment) as a surrogate measure of caspase-3-induced DFF40 endonuclease activity. (C) Quantitative ELISA determining the levels of cleaved-DFF45 (p11 fragment), expressed as experimental blank-corrected absorbance (O.D) measured at ?570 (570 nm). (D) Quantitative DFF40 endonuclease activity assay, expressed as experimental blank-corrected absorbances (O.D) measured at ?405 (405 nm). miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in Section 4.3) and are reported in Supplementary Figure S2A. The validation of the ectopic expression of the HA-tagged ca-GSK3ß-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in Supplementary Figure S2B). GSK3ß kinase activity (as described in Section 4.4) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca-GSK3ß-S9A mutant (reported in Supplementary Figure S2C). All data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4). ** p = 0.01; **** p = 0.0001; ns: not significant (p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation.