Fig. 5: 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/.