Fig. 3: SPD promotes endolysosomal degradation of GSDME-NT.
From: Methionine metabolite spermidine inhibits tumor pyroptosis by enhancing MYO6-mediated endocytosis
(A) Immunoblot of GFP-GSDME-NT in Dox-treated GSDME-NTTet-On HONE1 and HCT116 in response to Met deprivation with or without SPD supplement. (B) Transcriptional level of GFP-GSDME-NT detected by qPCR in Dox-treated GSDME-NTTet-On HONE1 and HCT116 in response to Met deprivation with or without SPD supplement. (C) Immunoblot of GFP-GSDME-NT in Dox-treated GSDME-NTTet-On HONE1 and HCT116 in response to CHX treatment (100 μg/ml) for the indicated time points. (D) Quantification of GFP-GSDME-NT level shown in (C). (E) Immunoblot of GSDME-NT oligomer and monomer in Dox-treated GSDME-NTTet-On HONE1 in response to Met deprivation with or without SPD supplement. (F) Immunoblot of GFP-GSDME-NT in Dox-treated GSDME-NTTet-On HONE1 and HCT116 in response to Dyngo4a (30 μM), CQ (50 μM), Pitstop-2 (30 μM) or MG-132 (10 μM) with or without Met deprivation. (G and H) Effects of Dyngo-4a, CQ and Pitstop-2 on Dox-induced PI uptake (G) and LDH release (H) in GSDME-NTTet-On HONE1 and HCT116. (I) Immunofluorescence of GFP-GSDME-NT and EEA1 in Dox-treated GSDME-NTTet-On HONE1 in response to Met deprivation with or without SPD supplement. Scale bar: 10μm. (J) Quantification of colocalization of GFP-GSDME-NT with EEA1 (n = 30 cells) shown in (I). (K) Immunofluorescence of GFP-GSDME-NT and lysotracker in Dox-treated GSDME-NTTet-On HONE1 in response to Met deprivation with or without SPD supplement. Scale bar: 10μm. (L) Quantification of colocalization of GFP-GSDME-NT with lysotracker (n = 30 cells) shown in (K). (M) Immunoblot of GFP-GSDME-NT of isolated endosomes and plasma membrane in Dox-treated GSDME-NTTet-On HONE1 in response to Met deprivation with or without SPD supplement. WCL, whole cell lysates; PM, plasma membrane. Data are represented as mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test (B, D, J, L); Two-way ANOVA with Bonferroni’s multiple comparisons test (G and H). NS, not significant. The results are representative of three independent experiments (A–M). Source data are provided as a Source Data file.
