Fig 1: PRKAB1 or PRKAB2 is required for the effect of MßCD on cholesterol reduction, autophagy induction, and increase of autophagy flux. (A, B) Filipin staining of NPC1 fibroblasts treated with MßCD. (A) PRKAB1 or PRKAB2 expression was silenced by shRNA and the subunits re-expressed with transient transfection of either PRKAB1 or PRKAB2 activation vectors. (B) ATG12 expression was silenced by shRNA. All of the cells were treated with 100 µM MßCD or DMSO control for 4 d followed by the filipin staining assay. (C) NPC1 fibroblasts transfected with ATG12 shRNA or control shRNA were treated with 100 µM MßCD or DMSO control for 24 h, followed by western blot analysis with the indicated antibodies. LC3B-II, SQSTM1 and ATG12 expression were normalized to GAPDH expression. (D-G) MßCD effects on SNARE proteins interactions. (D) Immunoprecipitation and western blot analysis of 3 SNARE proteins (VAMP8, STX17 and SNAP29). WT fibroblasts with the PRKAB/AMPK ß-subunit silenced or with AMPK ß-subunit re-expression was treated with MßCD or DMSO for 24 h. Cells were lysed and directly immunoprecipitated with anti-VAMP8 antibody followed by western blot analysis with the indicated antibodies. (E) NPC1 and WT fibroblasts were treated with MßCD for the indicated times, followed by immunoprecipitation with anti-SNAP29 and western blot analysis with the indicated antibodies. (F) Immunofluorescence staining and colocalization of LC3 with VAMP8. Indicated WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles, were treated with 100 µM MßCD or DMSO for 24 h and stained with anti-VAMP8 antibody. The punctate structures of VAMP8 were colocalized with RFP-LC3 (yellow color in the merged images). Data represent mean ± SEM of 10 images. (G) NPC1 and WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles were treated with MßCD for the indicated times, followed by staining with anti-VAMP8 antibody. The colocalization of VAMP8 and RFP-LC3 puncta was analyzed as above. Abbreviations: PRKAB1 or PRKAB2 act., PRKAB1 or PRKAB2 activation vectors; Mock vec., mock vector; LY, lysosome; AP, autophagosome. Scale bar: 10 µm (yellow) and 1 µm (white).
Fig 2: The O-GlcNAcylation level of SNAP-29 is associated with autophagy activity induced by cisplatin. (A) Control and OGT-deficient SKOV3 cells were transfected with NC siRNA or SNAP-29 siRNA and then treated with cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were examined by western blotting. (B) OGT-deficient SKOV3 cells were transfected with mRFP-GFP-LC3 vector for 24 h and then transfected with NC siRNA or SNAP-29 siRNA. After culturing in cisplatin (5 µg/mL) for 24 h, cells were imaged with a confocal microscope. Representative images of fluorescent LC3 puncta are shown. *P < 0.05 compared with OGT sh group. Scale bar represent 10 µm. (C) SKOV3 cells stably expressing control shRNA or OGT shRNA were treated with cisplatin (5 µg/mL) for 24 h and then the expression of SNAP-29 was tested by western blotting. (D) After being treated with cisplatin (5 µg/mL) for 24 h, the control and OGT-deficient SKOV3 cell extracts were immunoprecipitated with anti-SNAP-29 and the resulting precipitants were immunoblotted against O-GlcNAc. Whole-cell lysates were tested for SNAP-29 and actin. (E) Control and OGT-deficient SKOV3 cells were transfected with SNAP-29-GFP and RFP-LC3 vector and then treated with cisplatin (5 µg/mL) for 24 h. Colocalization of SNAP-29 and LC3 was determined by confocal fluorescence microscopy. Scale bar represent 10 µm. The values are presented as mean ± SD (n = 3). **P < 0.01, *P < 0.05.
Fig 3: BORC promotes recruitment of HOPS to lysosomes and assembly of autophagic STX17 SNARE complex. (A) Confocal microscopy of WT, BORCS5-KO and BORCS5-rescue HeLa cells transiently transfected with plasmids encoding mCherry-VPS41 and ARL8B-GFP. Lysosomes were visualized by LAMP1 staining. Nuclei were stained with DAPI. Scale bar: 15 µm. (B) Lysates of HEK293T cells transfected with plasmids encoding mCherry-VPS41, FLAG-STX17 and either MYC-BORCS6, ARL8B-GFP or MYC-BORCS6+ARL8B-GFP were immunoprecipitated using antibody to the MYC epitope followed by immunoblotting with antibodies to the indicated proteins. (C) WT, ARL8B-KO and ARL8B-KO-ARL8A-KD HeLa cells were transfected with plasmids encoding FLAG-VPS41, FLAG-STX17 and MYC-BORCS6. Cells were lysed and immunoprecipitated with antibody to MYC and immunoblotted with antibodies to the indicated proteins. (D) WT and BORCS5-KO HeLa cells were transfected with plasmids encoding FLAG-VPS41, FLAG-STX17 and either GFP or ARL8B-GFP. Cells were lysed and immunoprecipitated with antibody to GFP and immunoblotted with antibodies to the indicated proteins. (E) WT and BORCS5-KO HeLa cells were transfected with plasmids encoding GFP or GFP-STX17. Cells were lysed and immunoprecipitated with antibody to GFP and immunoblotted for the indicated proteins. In (B to E), the positions of molecular mass markers (in kDa) are indicated at left. (F) Quantification of VAMP8, SNAP29 and VPS41 in immunoprecipitates (normalized to input) from experiments as in (E). Bars represent the mean ± SEM from 3 independent experiments. *P < 0.05, **P < 0.001, the unpaired Student t test.
Fig 4: O-GlcNAcylation of SNAP-29 regulates the interaction of SNAP-29 with Stx17 and VAMP8. (A) SKOV3 cells were transfected with SNAP-29 and SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The O-GlcNAcylation levels of SNAP-29 were tested by co-immunoprecipitation. (B) SKOV3 cells were transfected with SNAP-29-GFP, SNAP-29 (Mut)-GFP and RFP-LC3 vectors and then treated with cisplatin (5 µg/mL) for 24 h. Colocalization of SNAP-29 and LC3 was determined by confocal fluorescence microscopy. Scale bar represent 10 µm. (C) SKOV3 cells were transfected with control, SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The expressions of LC3 and p62 were measured by western blotting. (D) The expressions of LC3 and p62 in SKOV3 cells that were transfected with si Stx17 or si VAMP8 and treated with cisplatin (5 µg/mL) for 24 h. (E) SKOV3 cell were transfected with SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. Then, cell extracts were immunoprecipitated with anti-SNAP-29 and the resulting precipitants were immunoblotted against Stx17 and VAMP8. Whole-cell lysates were tested for SNAP-29 and actin. The values are presented as mean ± SD (n = 3). **P < 0.01, *P < 0.05.
Fig 5: Schematic representation of the regulation of autophagosome-lysosome fusion by BORC. In WT cells, BORC recruits ARL8 and HOPS to the lysosomal membrane. HOPS in turn interacts with LC3B and STX17 on the autophagosomal membrane, promoting assembly of the STX17-VAMP8-SNAP29 trans-SNARE complex and autophagosome-lysosome fusion. In BORC-KO cells, ARL8B and HOPS remain in the cytosol, impairing STX17-VAMP8-SNAP29 trans-SNARE complex assembly and autophagosome-lysosome fusion.
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