Fig 1: CASP2 induces BID cleavage to mediate mitochondria‐dependent pyroptosis A SH‐SY5Y cells were infected with HSV‐2 (MOI = 1) for the indicated time intervals or stimulated with TG (20 μM, 16 h). Lysates were immunoblotted for BID and vinculin. tBID, truncated BID.B, C SH‐SY5Y cells transfected with AAVS1 or BID gRNA‐Cas9 RNPs were infected with HSV‐2. Lysates were isolated 16 h post‐infection and immunoblotted for GSDME, CL‐CASP3, and CL‐PARP (B), and assayed for CASP3 activity (C).D, E SH‐SY5Y control and BID‐depleted cells were infected with HSV‐2 (MOI = 1). Supernatant and lysates were evaluated for LDH (24 h) and HMGB1 (16 h) release. Vinculin blot panel E is identical to the one in panel B.F SH‐SY5Y control and CASP2‐depleted cells were infected with HSV‐2 (MOI = 1, 16 h) and immunoblotted for truncated BID, tBID.G NADPH reductase activity was measured to evaluate mitochondria function in SH‐SY5Y cells infected with HSV‐2 (MOI, 1) for the indicated time intervals.H–J SH‐SY5Y cells were treated with ethidium bromide (2 μg/ml) and infected with HSV‐2 (MOI = 1, 16 h). Lysate and supernatant were for immunoblotting for cell death markers GSDME and HMGB1 (H, I). SH‐SY5Y cells were transfected with Poly I:C (50 μg/ml) or dsDNA (20 μg/ml) for 24 h (J). Data information: All data shown are representative of at least three independent experiments. Data are presented as mean ± s.d. in all graphs. ***P ≤ 0.001 (Mann–Whitney test, two‐tailed in G, two‐way ANOVA in C and D). Source data are available online for this figure.
Fig 2: HSV‐2‐induced ER stress occurs upstream of pyroptosis A, B SH‐SY5Y cells were infected with HSV‐2 (MOI = 1) for the indicated time intervals, and immunoblotted for ER stress markers (BIP, phospho‐IRE1α, phospho‐eIF2α, cleaved‐ATF6, and CHOP) (A), and analyzed for mRNA levels of the ER stress response genes BIP, ATF6, and CHOP by RT–qPCR (B).C Lysate from SH‐SY5Y cells with HSV‐2 infection (MOI = 1) were immunoblotted for BCL2 family proteins MCL‐1, Bcl‐XL, and phospho‐BAD (S112). Vinculin was used as loading control.D–F SH‐SY5Y cells were stimulated with thapsigargin (TG, 20 μM, 24 h) and cycloheximide (CHX, 10 μg/ml, 24 h) and evaluated for LDH release (D) and immunoblotted for GSDME cleavage (E, F).G–K SH‐SY5Y cells were pretreated with TUDCA (500 ng/ml) for 1 h and infected with HSV‐2 (MOI = 1). Lysates and supernatant were isolated after 16 h of HSV‐2 infection and subjected to immunoblotting for CL‐CASP3, CL‐PARP, GSDME, HMGB1 (G, I), and tBID (K). The lysates (16 h) were also assayed for CASP2 activity (J), and supernatants (24 h) were analyzed for LDH release (H, ELISA).L SH‐SY5Y cells transduced with Lenti‐plasmid BIP or empty vector control were infected with HSV‐2 (MOI = 1, 16 h). Lysates were isolated and immunoblotted for CL‐CASP3, CL‐PARP, GSDME, and tBID. Data information: All data shown are representative of at least three independent experiments. Data are presented as mean ± s.d. in all graphs. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (Mann–Whitney test, two‐tailed in B, D, and J two‐way ANOVA in H). Source data are available online for this figure.
Fig 3: HSV‐2‐induced pyroptotic cell death in SH‐SY5Y cells depends on CASP2 A Lysates from SH‐SY5Y and THP1 cells were immunoblotted for a panel of caspases and vinculin.B SH‐SY5Y cells were pretreated with CASP2 inhibitor Z‐VDVAD (20 μM) for 1 h and infected with HSV‐2 (MOI = 1). Lysates were isolated 16 hpi for immunoblot analysis.C, D SH‐SY5Y cells were transfected with CASP2 gRNA‐Cas9 RNPs or AAVS1 gRNA‐Cas9 RNPs and infected with HSV‐2. Lysates were isolated 16 h post‐infection and assayed for CASP3 activity (D, ELISA) and immunoblotted for GSDME, CASP2, cleaved caspase 3 (CL‐CASP3), cleaved PARP (CL‐PARP), and vinculin (C).E SH‐SY5Y cells were infected with HSV‐2 (MOI = 1) for the indicated time points and lysates were analyzed for CASP2 activity.F, G SH‐SY5Y cells were infected with HSV‐2. Supernatant and lysates were isolated for measurement of LDH (F, 22 h) and identification of HMGB1 (G, 16 h).H SH‐SY5Y cells transfected with AAVS1 or CASP2 gRNA‐Cas9 RNPs and infected with HSV‐2 for 16 h and examined for morphological changes by microscopy. Boxes indicate areas highlighted in the zoomed images. Arrows indicate ballooning or syncytial cells. Data information: All data shown are representative of at least three independent experiments. Data are presented as mean ± s.d. in all graphs. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (Mann–Whitney test, two‐tailed in D, E, and F). Source data are available online for this figure.
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