Fig. 5: Lysosomal inhibition leads to accumulation of necroptosis mediators and sensitization to necroptosis in vitro.

a Accumulation of RIPK1 and RIPK3 in PC12 cells following treatment with lysosomal inhibitor chloroquine (Chq, 100 μM). Cells were treated for 4 h. Full unedited western blots are presented in Supplemental Figure S6a-b. b Quantification of RIPK1 and RIPK3+/− Chq treatment data from a. n = 14–20. c Accumulation of RIPK1 and RIPK3 in rat cortical neurons following treatment with Chq or Bafilomycin A (BafA, 100 nM). Cells were treated for 4 h. Full unedited western blots are presented in Supplemental Figure S6d. d Quantification of RIPK1 and RIPK3+/− Chq treatment data from c. n = 12. e Quantification of RIPK1 and RIPK3+/− BafA treatment data from c. n = 8. f IF staining for RIPK1 in control and Chq treated rat cortical neurons. Cells were treated for 4 h; LAMP1 was used to visualize lysosomes. Images were acquired at ×20. g Quantification of RIPK1 intensity from f. n = 12. h Quantification of RIPK3 intensity in rat cortical neurons with and without Chq treatment. n = 8–9 Representative images are in Supplemental Figure S6e. i Close-up of the areas indicated in f showing colocalization of LAMP1 and RIPK1. Arrows point to RIPK1 positive lysosomes. j Quantification of RIPK1 positive lysosomes from i. n = 12. k Potentiation of necroptosis in PC12 cells treated with lysosomal inhibitor BafA. PC12 cells were treated with cycloheximide (20 μg/ml), pan-caspase inhibitor Boc-D (50 μM), and indicated doses of rat TNFα (0, 25, 50 ng/ml) to induce necroptosis in the presence or absence of BafA (100 nM). RIPK1 inhibitor necrostatin 1 (Nec1, 30 μM) was used to confirm that cell death was dependent on necroptosis. After 18 h cell viability was measured using luminescent ATP assay. n = 6 Additional controls are presented Supplemental Figure S7. l Data from k presented as % cell death. All data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001