Fig. 5: The lysoptosis-like cell death pathway involves promiscuous cytoplasmic proteolytic activity.
From: Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins
a Western blot analysis of lysosomal CTSL (goat polyclonal cathepsin L, Novus, AF952), nucleocytoplasmic SERPINB3 (α-B3; rabbit polyclonal SERPINB3/B4), and cytosolic GAPDH () in supernatants of HT3B3-WT and HT3B3-KO cell lines treated with 100 or 10% DPBS for 30 min at 37 °C, 5% CO2 prior to treatment with DPBS containing 0, 20, or 200 µg/ml digitonin. The relative band intensities (rel. int.) for each lane were calculated (Image Lab, v6.1, Bio-Rad) and are shown under the corresponding lane. Note the detection of CTSL in the 20 µg/ml digitonin supernatants of both HT3B3-WT and HT3B3-KO treated with 10% DPBS but not the 100% DPBS controls. Black arrowheads indicate full-length proteins, gray and open arrowheads demark processed active forms of CTSL. b Pan-lysosomal cysteine protease activity (zFR-R110). Activity in supernatants of HT3B3-WT and HT3B3-KO cell lines treated with 100 or 10% DPBS for 30 min at 37 °C, 5% CO2 prior to treatment with DPBS containing 0, 20, or 200 µg/ml digitonin. The activity was normalized to the 200 µg/ml control to account for total lysosomal cysteine protease protein levels. Note, the significantly enhanced cysteine protease activity in HT3B3-KO cytosolic supernatants (20 µg/ml digitonin) compared to HT3B3-WT in 10% DPBS cells treatment. c Bulk CTSL-KO SW756B3-WT or SW756B3-KO cell lines were generated by CRISPR/cas9 methodology. The percentage of the bulk population containing indels in the CTSL gene was confirmed by NGS sequencing (Table S1) and CTSL protein levels are shown in ref. 114. Cell lines were then treated with either 100 or 10% DPBS in the presence of SG for 4 h. The % dead was calculated as the number of SG positive cells/the total number of cells as determined by high contrast brightfield imaging × 100. Analyses were compared using a two-way ANOVA with Tukeys’ multiple comparisons (n ≥ 10 replicates; *** P < 0.001). Note, the statistically significant reduction in % dead between SW756B3-KO;CTSL-WT and SW756B3-KO;CTSL-KO. d HT3B3-WT or HT3B3-KO cells were incubated with DMSO, 10 µm E64d, or 10 µm cathepsin L selective inhibitor, CAA0225 for 1 h prior to exposure to 100 or 10% DPBS for 14 h, and stained with SG. Percent dead was calculated as ((Sytox positive nuclei/total cells calculated by brightfield microscopy) × 100) (n = 5 replicates; ns not significant, *** P < 0.001). Uncropped immunoblots can be found in Supplementary Fig. 21.
