Fig. 3: RIP3 is dispensable in FGFR2 inhibition-induced necroptosis. | Nature Communications

Fig. 3: RIP3 is dispensable in FGFR2 inhibition-induced necroptosis.

From: Harnessing the FGFR2/NF2/YAP signaling-dependent necroptosis to develop an FGFR2/IL-8 dual blockade therapeutic strategy

Fig. 3

a Western blot analysis of RIP1 and RIP3 expression of esophageal cell lines. b Schematic of the molecules participated in necroptosis under various conditions. Created in BioRender. Chen, D. (2025) https://BioRender.com/ukteuom. c Transmission electron microscopy of the COLO680N cells treated for 12 h with DMSO or 10 μM AZD4547. Yellow arrowheads denote loosened nuclei. Red arrowheads indicate swollen cellular organelles. Blue arrowheads show plasma membrane rupture. Scale bar: 2 μm. d Following transfection with RIP1, RIP3 or MLKL siRNA for 48 h, cell viability was determined after treatment with AZD4547 for another 24 h. The p value was derived from a comparison with the control group. e Cells were treated with 40 μM AZD4547 ± 30 μM Nec-1, 5 μM NSA, 20 μM ZVAD and 1 μM Fer-1 for 24 h to assay cell viability. f Cells were transfected with RIP3 siRNAs for 48 h following AZD4547 treatment for another 24 h and membrane-cytosolic extraction was analyzed by immunoblotting. g Immunoprecipitation assay for MLKL binding to RIP1 and RIP3 post-AZD4547 treatment (40 μM). h Indicated L929s were treated with 10 μM AZD4547 for 24 h, and then cell viability was assayed. The p value was derived from a comparison with the WT group. i Immunoblotting of indicated proteins in L929 RIP3−/− cells following treatment with AZD4547 (0.1–10 μM) for 24 h. j Cells were treated with 1 μM AZD4547 for 24 h and indicated proteins of membrane-cytosolic extraction were analyzed by immunoblotting. k Following transfection with RIP1 and MLKL siRNA for 48 h, 10 μM AZD4547 was added for subsequent 24 h to assay cell viability. The p value was derived from a comparison with the control group. l Cells were treated with AZD4547 with or without Nec-1, NSA, ZVAD, and Fer-1 for 24 h to assay cell viability. ns not significant. All data are mean ± SD from n = 4 (k, l) or 5 (d, e, h) biological replicates. Statistical analyses were performed using one-way ANOVA with multiple comparisons. Source data are provided as a Source Data file.

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