Fig 2: Pyroptosis might not be promoted by SARS-CoV-2 infection in HMC3. (A) After SARS-CoV-2 infection in HMC3, NLRP3, GSDMD, and caspase-1 proteins were analyzed by Western blotting. Hsp70 served as a loading control. (B) The SARS-CoV-2-infected HMC3 cells were treated with caspase-1 inhibitors (Ac-FLTD-CMK and Belnacasan) for 6 days at the indicated concentrations. The surviving cells were stained with crystal violet, and then the percent measurements of the stained cell covered areas were determined by the ImmunoSpot reader. Statistically significant differences between the groups were determined by one-way ANOVA; ****, P < 0.0001. Symbols represent means ± SEM.

Fig 3: FCA NP-loaded MET&DOX induces melanoma cell death via induction of pyroptosis, apoptosis, and necroptosis (PANoptosis). (A) Representative confocal microscopy images of cleaving mNeon-GSDMD expressed in human A375 and SK-MEI-28 cells, scale bars: 20 µm. (B) Apoptosis and necroptosis of human A375 and SK-MEL-28 cells following 24-h incubation with different formulations; n = 3 for each group. (C) The protein expression levels of the GSDMD, CASPASE-7, and MLKL protein in human A375 and SK-MEL-28 cells following 24 h incubation with different formulations; n = 4 for each group. (D) IHC analysis of the GSDMD, Caspase-7, and MLKL protein expression in the corresponding tumor tissues obtained after the different treatments; scale bars: 50 µm, n = 5 for each group. CTL: FCA NPs, MET FCA NPs: FCA NP-loaded MET, DOX FCA NPs: FCA NP-loaded DOX, MET&DOX FCA NPs: FCA NP-loaded MET&DOX.

Fig 4: Pin1 deficiency prevents caspase-1-mediated cleavage of gasdermin D (GSDMD). (A) Imaging assay of pyroptosis in WT and Pin1-/- bone marrow-derived macrophages (BMDMs) treated with 5 mM ATP or 20 mM nigericin. (B) BMDMs were primed with 500 ng/ml LPS for 4 h and stimulated with ATP or nigericin for 30 min and supernatants were analyzed for lactate dehydrogenase (LDH). (C) Immunoblotting with mouse antibody for GSDMD in WT and Pin1-/- BMDMs treated with ATP or nigericin. (D) Immunoblotting with mouse antibody for GSDMD in RAW 264.7 cells transfected with control siRNA or siRNA for Pin1 treated with 5 mM ATP or 20 mM nigericin for 30 min. All data are representative of three independent experiments. The data represent the mean ± SD of one among three biological replicates, with three technical replicates each. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance; two-way ANOVA (B).

Fig 5: GSDMD knockdown decreases the MMP of NSCLC cell lines. (A and B) MMP was detected by the JC-1 probe at 72 h post-transfection (NC, si-1031 or si-1244). Normal MMP cells are indicated by high green fluorescence (FL1) and red fluorescence (FL2), Low MMP cells are indicated by high FL1 and low FL2. (C and D) Statistical analysis of low MMP cell percentage. Data are represented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 (ANOVA test). MMP, mitochondrial membrane potential; GSDMD, gasdermin D; NSCLC, non-small cell lung cancer; NC, negative control.