Fig 2: RPE flatmounts are viable for at least one week after culture in vitro. (A) RPE flatmounts were prepared from 2- and 9-month-old wild-type C57BL/6J mice and cultured in complete medium with or without H2O2 for up to one week. Protein lysates were prepared from the RPE flatmounts and subjected to Western blotting analysis. The cell death marker RIP1 was used to evaluate cell viability; vinculin was used as an internal control. As a positive control: RPE flatmounts treated with either 1 mM or 10 mM H2O2 had significant expression of RIP1. Further, the RPE flatmount that was cultured for 7 days without changing the medium (mishandled sample) showed a slight amount of RIP1, whereas the RPE flatmounts cultured with frequent medium changes did not express RIP1, suggesting that the RPE cells obtained even from older mice (9 months) were viable after at least 7 days of culture. (B) RPE flatmounts were obtained from 3-month-old wild-type C57BL/6J mice and cultured in complete medium with or without H2O2 treatment for up to one week. RPE flatmounts were then fixed in 2.5% glutaraldehyde and processed for transmission electron microscopy (TEM). The TEM imaging of the RPE flatmounts cultured for 16 h, 3 days, and 7 days showed that these RPE cells have normal nuclei (yellow arrows) and organelles, such as mitochondria (yellow arrowheads), and abundant microvilli. H2O2-treated RPE flatmounts showed cell death symptoms, including chromatin condensation and fragmentation (red arrow), a disrupted cell membrane, complete loss of microvilli, a translucent cytoplasm, and severely damaged mitochondria (red arrowheads). (C) ZO-1 immunostaining on 2-month-old RPE flatmounts cultured for 3 days showed robust ZO-1 expression, indicating RPE cell integrity.

Fig 3: ABT-737 induces cancer cell necrosis and inhibits cell viability and invasion when the RIP1 gene is knocked down in bladder cancer cells. (A) Expression levels of RIP1 in UMUC3 and 5637 cells were analyzed via western blotting after treatment with RIP1 siRNA for 12 h, and the results were (B) semi-quantified. (C) Protein expression levels of HMGB1, ZBP1, RIP3 and MLKL were analyzed in RIP1-KD UMUC3 and RIP1-KD 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. The results were semi-quantified in (D) UMUC3 and (E) 5637 cells. (F) Transwell invasion assays were performed with RIP1-KD UMUC3 cells. (G) Quantification of Transwell assay results (H) MTT assays were performed to examine the viability of RIP1-KD UMUC3 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. (I) Transwell invasion assays were performed with RIP1-KD 5637 cells. (J) Quantification of Transwell assay results. (K) MTT assays were performed to examine the viability of RIP1-KD 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. *P<0.05 vs. control or siRNA NC. OD, optical density; scale bar=50 µm; ZBP1, Z-DNA binding protein 1; RIP, receptor-interacting protein; HMGB1, high mobility group box 1; MLKL, mixed-lineage kinase domain-like protein; siRNA, small interfering RNA; NC, negative control; KD, knockdown.

Fig 4: Co-IP results and a schematic diagram of the mechanism underlying ABT-737 upregulation of the interaction of ZBP1 with RIP3 in inducing bladder cancer cell necrosis. (A) Co-IP was performed using anti-RIP3 antibody with UMUC3 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. (B) IP was performed using anti-RIP3 antibody with 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. (C) A schematic diagram showing the mechanisms via which ABT-737 induced necrosis. IP, immunoprecipitation; ZBP1, Z-DNA binding protein 1; RIP, receptor-interacting protein; MLKL, mixed-lineage kinase domain-like protein; Cyt-C, cytochrome C.

Fig 5: Nec-1 treatment inhibits necroptosis induced by CoCl2 in C2C12 cells. (A) Western blot analysis revealed the protein levels in C2C12 myotubes treated in the absence or presence of CoCl2 and Nec-1. Band intensity was quantified using ImageJ software. Tubulin was used as an internal control. *P<0.05, compared with the control group; #P<0.05, compared to the CoCl2 group. (B) Transmission electron microscopic images of C2C12 cells. Scale bar=2 µm. CoCl2, cobalt chloride; RIP1, receptor-interacting protein 1; RIP3, receptor-interacting protein 3.