Fig 2: TF-3 attenuates emphysema in CSE-treated mice. (A) Images of lung tissue stained with H&E. Scale bars, 20 µm. (B and C) Morphometric measurements of the MLI and DI in the four groups were quantified. (D and E) TNF-α and IL-1β levels in lung tissue were detected using ELISA and quantified. (F-H) MDA, SOD and GSH levels in lung tissue were detected with assay kits and quantified. (I) Protein expression levels of p-RIPK3/RIPK3 and p-MLKL/MLKL were determined via western blotting with GAPDH as an internal control. Semi-quantification of protein expression levels of (J) p-RIPK3 and (K) p-MLKL were determined. Data are presented as the mean ± SEM of five independent experiments indicated by dots. One-way ANOVA was performed followed by Tukey's post hoc test for statistical comparisons among more than two groups. *P<0.05, **P<0.01 and ****P<0.0001. MLI, mean linear intercept; DI, destructive index; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; RIPK3, receptor-interacting serine/threonine-protein kinase 3; MLKL, mixed lineage kinase domain-like; p, phosphorylated; CSE, cigarette smoke extract; TF-3, theaflavin-3,3'-digallate.

Fig 3: HtrA2 enhanced necroptosis by degrading RIPK1.a HT-29 cells were pretreated with UCF-101 (50 μM) for 1 h, followed by stimulation with T/S/Z for different times as indicated. Phosphorylation of RIPK1, RIPK3, and MLKL, as well as their protein levels, were analyzed by immunoblotting with corresponding antibodies. b Silencing of HtrA2 inhibited formation of necrosome in T/S/Z treated HT-29 cells. HT-29-shNC or HT-29-shHtrA2 cells were stimulated with T/S/Z for indicated times. The association between RIPK1 and RIPK3 was analyzed by immunoprecipitation with anti-RIPK1 antibody, followed by immunoblotting. c HT-29 cells stably expressing HtrA2-3× Flag fusion protein were stimulated with T/S/Z for indicated times. The association between RIPK1 and HtrA2-3× Flag was analyzed by immunoprecipitation with anti-Flag antibody, followed by immunoblotting. d HtrA2 translocated from mitochondria to cytosol during necroptosis. HT-29 cells were stimulated with T/S/Z for different times as indicated. Thereafter, the cytosol and the mitochondrial fractions were subjected to SDS-PAGE. Immunoblotting was performed with specific antibodies for the indicated proteins. e HtrA2 translocated from mitochondria to cytosol from mitochondria to cytosol during DSS-induced colitis. The cytosol and the mitochondrial fractions of colon tissues from normal mice (day 0) and DSS-treated mice (day 7) were subjected to SDS-PAGE. Immunoblotting was performed with specific antibodies for the indicated proteins. In d, e, antibodies to TOM20 and GAPDH served as controls for purity of mitochondrial and cytosolic fractions, respectively

Fig 4: Cell death in B3-KO cells following RT suggests potential engagement of multiple cell death mechanisms including apoptosis/caspase-dependent cell death and lysosome-dependent necrosis with features of lysoptosis.a Western blot of total cell lysates from HT3-B3-WT, -B3-KO cells at indicated time points following sham or increasing doses of RT. Positive control lysates from HT3-B3-WT cells treated with etoposide (A for apoptosis), or nucleofected LPS (P for pyroptosis), and HT29 cells treated with TNFα, BV6 and ZVAD-fmk (N for necroptosis) are shown in the center of the blot. Relative intensity of cleaved-caspase-3 (CC-3) and cleaved-caspase-7 (CC-7) is shown normalized to intensity of actin bands, using the positive control for apoptosis as reference. Histogram shows % Sytox-positive cells for each condition determined in parallel (bar = mean of triplicate wells, 2–4 fields of view per well, error bars = standard deviations). Full length GSDMD indicated by black asterisk, and p30 cleavage product indicated by black arrow. Phospho-RIPK3 band (~60 kDa) is indicated by a red arrow, ~75 kDa band on SW756 blot (indicated by red asterisk) is residual PARP antibody signal from previous blotting. b Western blot of total cell lysates from SW756-B3-WT, -B3-KO cells at indicated time points following sham or increasing doses of RT. Positive control lysates from HT3-B3-WT cells treated with etoposide (A for apoptosis), or nucleofected LPS (P for pyroptosis), and HT29 cells treated with TNFα, BV6 and ZVAD-fmk (N for necroptosis) are shown in the center of the blot. These positive control lysates were generated concurrently with the experiment. Histogram shows % Sytox-positive cells for each condition determined in parallel. c Representative light field images of SA-βgal stained SW756-B3-WT or B3-KO cells 96 h after treatment with sham or 30 Gy, scale bars = 50 μm. d Quantitation of percent positive X-gal staining cells with individual data points, mean and standard deviation. Statistical tests are t-test comparisons except ANOVA as noted, * = p < 0.05, n.s. = not significant. e WB of total cell lysates from HT3-B3-WT or -B3-KO or SW756-B3-WT or –B3-KO cells treated as indicated. Blots were probed with anti-BCL-2, anti-BAX and GAPDH as a loading control, with quantified band intensity below, normalized to GAPDH. f Percent Sytox-positive HT3-B3-WT or –B3-KO cells (left) or SW756-B3-WT or –B3-KO cells (right) treated with indicated pharmacologic inhibitors 24 h after sham or radiation. (bar = mean of triplicate wells, 2–4 fields of view per well, error bars = standard deviations, individual data points shown). g Percent TUNEL + nuclei / total nuclei determined on TUNEL stained formalin fixed paraffin embedded sections from indicated tumors (an entire tissue section was analyzed for n = 2 mice per group, n = 3 for B3-WT sham). Bar height indicates mean. h–k representative images of TUNEL staining. Scale bar = 40 µm. l Histogram of percent cleaved-caspase-3 (CC-3) + nuclei / total nuclei determined on IHC with CC-3 antibody stained from indicated tumors (an entire tissue section was analyzed for n = 2 mice per group, n = 3 for B3-WT sham). Bar height indicates mean, * = p < 0.05, n.s. = not significant. m–p representative images of CC-3 staining. Scale bar = 40 µm. Full slide digital images are available at: https://app.histowiz.com/shared_orders/2ccb0ce8-e4e1-41d1-8f75-a0e621ced36e/slides/.

Fig 5: Necroptotic cell death is not activated by SFN 50 μM. Evaluation by Western Blot of phosphorylated protein levels for MLKL (p-MLKL; a) and RIP3 (p-RIP3; b) in U-937 cells treated with SFN 50 μM for the indicated time. U-937 cells treated with 50 ng/mL TNF-α + 500 nM SM-164 (S) + 50 µM Z-VAD-FMK (TSZ) for 4 or 6 h were used as positive control for RIP3 induction and MLKL phosphorylation. Upper panel shows representative Western Blot results of p-MLKL (a) and p-RIP3 (b); lower panel shows protein quantification expressed as relative levels of p-MLKL (a) and p-RIP3 (b) normalized to β-actin.