Fig 2: GSDME knockdown promotes the degradation of EGFR. A Western blot analysis of EGFR and GSDME levels in A549 and H1299 cells treated with 20 μg/mL cycloheximide (CHX) for 0 h, 6 h, 12 h and 24 h after transfection with siScr or siGSDME for 24 h. β-Actin was used as the loading control. B Western blot analysis of EGFR and GSDME levels in H157 cells treated with 15 μM E64D or 20 μM MG132 for 6 h after transfection with siScr or siGSDME for 24 h. GAPDH was used as the loading control. C EGFR ubiquitination was detected by a co-IP assay in GSDME-depleted H157 cells. H157 cells were transfected with pcDNA3.1-HA-EGFR, pcDNA3.1-HIS-Ub, siScr or siGSDME for 24 h and then treated with 15 μM E64D, 20 μM MG132 for 6 h and 100 ng/mL EGF for 15 min. A co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with HIS, HA and GSDME antibodies. The numbers shown above the bands were fold changes of band intensities relative to the control. The band intensities of HIS were quantified by Image J software and normalized to HA. The band intensity of HIS was relatively increased in GSDME-knockdown H157 cells. GAPDH was used as the loading control for the input. D Up: Immunofluorescence of EGFR (red fluorescence) and EEA1 (green fluorescence) in GSDME-depleted A549 cells. Scale bar, 10 μm. Down: quantification of the co-localization of EEA1 and EGFR. RNAi effency of A549 cells were measured by Western blot analysis. β-Actin was used as the loading control. E The interaction of EGFR and c-Cbl was detected by a co-IP assay in GSDME-depleted H1792 cells. Cells were transfected with pcDNA3.1-HA-EGFR, siScr or siGSDME for 24 h and then treated with 15 μM E64D, 20 μM MG132 for 6 h and 5 ng/mL EGF for 10 min. A Co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with c-Cbl, HA and GSDME antibodies. The numbers shown above the bands were fold changes of band intensities relative to the control. The band intensity of c-Cbl was quantified by Image J software and normalized to HA. c-Cbl level was relatively increased in GSDME knockdown H1792 cells. β-Actin was used as the loading control of the input. F Western blot analysis of phosphorylated EGFR (Y1045), EGFR and GSDME levels in A549 cells treated with 15 μM E64D or 20 μM MG132 for 6 h and 10 ng/mL EGF for 0 min, 10 min, 20 min, 40 min and 80 min after transfection with siScr or siGSDME for 24 h. G Western blot analysis of phosphorylated EGFR (Y1045), EGFR and GSDME levels in H157 cells treated with 15 μM E64D or 20 μM MG132 for 6 h and 10 ng/mL EGF for 0 min, 10 min, 20 min and 40 min after transfection with siScr or siGSDME for 24 h. β-Actin was used as the loading control

Fig 3: Early endosomes are enlarged and accumulate brain-derived neurotrophic factor (BDNF) and tropomyosin kinase B (TrkB) in spinocerebellar ataxia type 6 (SCA6) Purkinje cells.(a) Schematic showing BDNF and TrkB endocytosis and trafficking to early endosomes labeled with early endosome antigen 1 (EEA1). (b) The early endosome marker EEA1 stains early endosomes in lobule 3 of the cerebellar vermis in wildtype (WT) and SCA6 mice, calbindin labels Purkinje cells. Scale bar, 100 µm. (c) Closeup images of the early endosome marker EEA1 in lobule 3 of cerebellum, calbindin labels Purkinje cells (outlined). Scale bar, 10 µm. (d) The area covered by EEA1 staining in Purkinje cells is increased in SCA6 mice compared to WT (p < 0.001; N = 6 WT mice, 173 cells; 5 SCA6 mice, 171 cells). (e) Representative images of the early endosome marker EEA1 and BDNF within Purkinje cells (outlined) in the anterior vermis. Scale bar, 10 µm. (f) Relative staining level of BDNF within early endosome compartment is higher in the Purkinje cells of SCA6 mice compared to WT (p < 0.001; N = 6 WT mice, 173 cells; 5 SCA6 mice, 171 cells). (g) Representative images of the early endosome marker EEA1 and TrkB within Purkinje cells (outlined) in the anterior vermis. Arrowheads denote locations of significant TrkB accumulations. Scale bar, 10 µm. (h) Relative staining level of TrkB within early endosome compartment is higher in the Purkinje cells of SCA6 mice compared to WT (p = 0.013; N = 4 WT mice, 79 cells; 4 SCA6 mice, 58 cells). Mann–Whitney U-test used for all statistical comparisons, *p < 0.05, ***p < 0.001.

Fig 4: Brain-derived neurotrophic factor (BDNF) immunostaining in the cerebellum requires heat-induced epitope retrieval (HIER) and is not detectable in the majority of liver tissue.(a) HIER greatly enhances the signal from both BDNF and early endosome antigen 1 (EEA1) staining in cerebellar vermis tissue from wildtype (WT) and spinocerebellar ataxia type 6 (SCA6) mice. Calbindin staining is unaffected. Scale bars, 10 µm. (b) BDNF staining is undetectable in the majority of liver tissue from WT mouse, with only the BDNF-positive putative cholangiocytes (arrowheads) showing immunoreactivity with the BDNF antibody. Background slice was incubated without primary antibody but all other staining steps proceeded as normal. Scale bars, 20 µm.

Fig 5: Tropomyosin kinase B (TrkB) activation with 7,8-dihydroxyflavone (7,8-DHF) rescues early endosomes deficits in spinocerebellar ataxia type 6 (SCA6) Purkinje cells.(a) Schematic showing 7,8-DHF administration to SCA6 mice and the previously described therapeutic benefits of 7,8-DHF in SCA6 mice. (b) Representative images of the early endosome marker EEA1 and brain-derived neurotrophic factor (BDNF) within Purkinje cells (outlined) of the anterior vermis. Scale bar, 10 µm. (c) The area covered by EEA1 staining in Purkinje cells is decreased in SCA6 mice given DHF compared to those that received control water (p < 0.001; Student’s t-test; N = 3 SCA6 mice, 50 cells; 3 SCA6 DHF mice, 63 cells). (d) Relative staining level of BDNF within early endosome compartment is decreased in the Purkinje cells of SCA6 mice that received DHF compared to controls (p < 0.001; Mann–Whitney U-test; N = 3 SCA6 mice, 50 cells; 3 SCA6 DHF mice, 63 cells). ***p < 0.001.