Fig. 4: SPD directly interacts with MYO6 to regulate endocytosis of GSDME-NT.
From: Methionine metabolite spermidine inhibits tumor pyroptosis by enhancing MYO6-mediated endocytosis
(A) Schematic depicting the production of SPD FG bead. (B) Venn diagram displaying the intersection among SPD-binding proteins and GSDME-NT-binding proteins (cells fed with CM or Met-omitted medium) in Dox-treated GSDME-NTTet-On HONE1. (C) Immunoblot of GFP-GSDME-NT in Dox-treated GSDME-NTTet-On HONE1 and HCT116 with MYO6 knockdown. (D and E) Effects of MYO6 knockdown on Dox-induced PI uptake (D) and LDH release (E) in GSDME-NTTet-On HONE1 and HCT116. (F) Immunofluorescence of GFP-GSDME-NT and EEA1 in Dox-treated GSDME-NTTet-On HONE1 with MYO6 knockdown. Scale bar: 10μm. (G) Quantification of colocalization of GFP-GSDME-NT with EEA1 (n = 30 cells) shown in (F). (H) Immunoblot of GFP-GSDME-NT of isolated endosomes and plasma membrane in Dox-treated GSDME-NTTet-On HONE1 with MYO6 knockdown. WCL, whole cell lysates; PM, plasma membrane. (I) High concentration of SPD (2 mM) addition to lysates from Dox-treated GSDME-NTTet-On HONE1 inhibited capture of MYO6 proteins by SPD-conjugated beads. (J) CETSA displaying an increase thermal stability of MYO6 by SPD addition. (K) In situ Duolink-PLA assay displaying the molecular interaction between SPD and MYO6 in Dox-treated GSDME-NTTet-On HONE1. Scale bar: 10μm. (L) Quantification of PLA signal per cell (n = 30 cells) shown in (K). (M) BLI analysis of the molecular binding between SPD and MYO6. Data are represented as mean ± SD. Two-way ANOVA with Bonferroni’s multiple comparisons test (D and E); Two-tailed Student’s t test (G and L). NS, not significant. The results are representative of three independent experiments (C–L). Source data are provided as a Source Data file.
