Fig. 5

DCBLD1 inhibits the autophagic degradation of G6PD. A HeLa cells expressing both the vector and DCBLD1 were treated with 10 μg/ml CHX for the specified durations. Whole-cell extracts (WCE) were collected for immunoblotting to detect G6PD levels in the cells. GAPDH was used as a loading control. B HeLa and C33A cells expressing shDCBLD1 were treated with MG132 (10 μm), BafA1 (200 nM), and CQ (25 μm) for 24 h. WCE were collected for immunoblotting to detect G6PD in the cells. GAPDH was used as a loading control. C, D HeLa and C33A cells expressing shCtrl and shDCBLD1, and WCE were collected for immunoblotting to detect LC3 I, LC3 II, P62, and mTOR/P-mTOR levels in the cells. GAPDH and Tubulin were used as loading controls. E Tumor sections from the xenograft tumor model were stained with IHC for LC3. F A statistical plot illustrating metabolite changes in HeLa cells expressing shDCBLD1 than those expressing shCtrl. G HeLa and C33A cells expressing shCtrl and shDCBLD1 were cultured under conditions of glucose deprivation and treated with Rapamycin (25 nM) for 24 h. LC3 puncta formation was subsequently detected through immunostaining. Scale bar: 1 μm. H Representative confocal images of GFP-LC3 and mRFP-LC3 distribution in HeLa and C33A cells transfected with DCBLD1 shRNA vector, both with or without rapamycin treatment and glucose deprivation for 24 h. Scale bar: 5 μm. I Representative images illustrating the autophagic structures in HeLa and C33A cells transfected with the DCBLD1 shRNA vector, both with or without CQ treatment for 24 h (25 μM). Scale bar: 2 μm, 1 μm, and 0.5 μm