Fig 2: Representative photomicrographs of immunohistochemistry for 8-OHdG (A), urine 8-OHdG concentration (B), immunoblotting analysis of MnSOD and NOX-4 (C), and intracellular ROS production in H2O2-treated HK-2 cells with or without Dapa and RIP inhibitors (D). Data are presented as mean ± SEM (in vivo, n = 6; in vitro, n = 3) and analyzed by one-way ANOVA. *** p < 0.01 vs sham or control; # p < 0.05 vs. UUO or H2O2.

Fig 3: Representative photomicrographs of gross finding (A), HE staining (B), transmission electron microscopy (C), and immunoblotting analysis of RIP1-RIP3-MLKL protein in vivo (D) and in vitro (E). UUO induces a crowd of necrosis within the tubular epithelium (circle) and abscission of microvilli in epithelial cells (arrows), whereas these are improved following administration of Dapa or RIP inhibitor. Data are presented as mean ± SEM (in vivo, n = 6; in vitro, n = 3) and analyzed by one-way ANOVA. *** p < 0.01 vs sham or control; # p < 0.05 vs UUO or H2O2.

Fig 4: Stress granule inducers inhibit RIPK1, RIPK3, and MLKL phosphorylation.Necroptosis signaling was analyzed by immunoblotting at the indicated time after the induction of necroptosis by TNF treatment. (A, B, C, D, E) Lysates from primary BMDMs pre-stressed with (A) thapsigargin (Thaps), (B) brefeldin A, (C) tunicamycin (Tunica), (D) MG132, or (E) Thaps and treated with zVAD + TNF were collected at the indicated time-points. Necroptosis signaling was examined by immunostaining for phosphorylation of MLKL and RIPK3 (p-MLKL and p-RIPK3) and phosphorylation of RIPK1 (p-RIPK1) at serine-166 (S166) or serine-321 (S321) and activation of the integrated stress response by phosphorylation of eIF2α (p-eIF2α). Immunoblots are representative of at least two independent experiments.

Fig 5: Representative photomicrographs of HE staining (A), transmission electron microscopy (B), and immunoblotting analysis of RIP1-RIP3-MLKL protein in vivo (C) and in vitro (D). UUO caused necrosis within the tubular epithelium (circle) and abscission of microvilli in epithelial cells (arrows), whereas these changes were attenuated by RIP inhibitor or ICG-001. ***P< 0.01 vs. sham or control; #P< 0.05 vs. UUO or H2O2.