Fig 2: The correlation between HMGB1 levels and the prognosis of GB patients treated with TMZ. A The mRNA levels of HMGB1 in non-tumor tissues (n = 14) and GB tissues (n = 528) from TCGA-GBM/HGU133 datasets (left panel), as well as in WHO II (n = 226), WHO III (n = 244) and WHO IV (n = 150) grade glioma tissues from TCGA-LGG/GBM datasets (right panel). B The overall survival (OS) rates of mRNA HMGB1low and HMGB1high GB patients from CGGA datasets (n = 489). C Representative immunohistochemistry (IHC) of HMGB1 in paired GB patients (left panel) with quantification (right panel). HMGB1 stains brown color. n = 41, Scale bars = 50 µm. D The concentration of HMGB1 in sera of paired GB patients. n = 41. E The OS rate of TMZ-treated serum HMGB1low and HMGB1high GB patients. n = 41. F Representative IHC of LC3B, SQSTM1, STX17 and HMGB1 in serial sections of GB specimens (GB7225 and GB1338) with TMZ treatment. Areas examined under higher magnification were marked. Brown color is positive. Scale bars = 50 µm. G The correlation between LC3B and HMGB1 expression in GB patients with TMZ treatment (n = 41) (upper panel). The correlation between SQSTM1 and HMGB1 expression in GB patients with TMZ treatment (n = 41) (lower panel). Pearson’s correlation test. *P < 0.05, **P < 0.01,***P < 0.001, ns = no significance

Fig 3: DDRGK1 deficiency results in lysosomal dysfunction.a Western blot analysis for SNAP29, Stx17, and VAMP8 proteins in EtOH- and 4-OHT-treated MEFs. b Quantitative expressions of SNAP29, Stx17, and VAMP8 proteins normalized to ß-actin. Data represent mean ± SEM; n = 3. ns not significant, P > 0.05. c The transcriptional levels of Snap29, Stx17, and Vamp8 in EtOH- and 4-OHT-induced MEFs. Data represent mean ± SEM; n = 3. *P < 0.05; ns not significant, P > 0.05. d Lyso-Tracker Red signals in control and DDRGK1-deleted MEFs. MEFs cultured in EtOH or 4-OHT for 4 days were then incubated with diluted Lyso-Tracker Red dye (diluted in cell culture medium in a ratio of 1:20,000) for another 20 min. Images were detected by laser confocal-scanning microscopy. Bar, 20 µm. e Quantification of normalized number of Lyso-Tracker Red puncta. Data were shown as mean ± SEM; n = 3. **P < 0.01. f Western blot analysis for CTSD expression in EtOH- and 4-OHT-induced MEFs. g Quantification of CTSD level normalized to ß-actin expression. Data represent mean ± SEM; n = 3. *P < 0.05.

Fig 4: TCHP contributes to autophagosome maturation The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X-100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3-h incubation with BafA1 (scale bars, 600 nm): (i), quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and (ii), distribution of the cross-section areas of the analysed vesicles expressed in percentage (n = 7 cells; > 100 vesicles per sample; AVs: unpaired t-test; **P < 0.0001 vs. control; APs: unpaired t-test **P < 0.0001 vs. control). Left panels: representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 and 2 µm in the inset. Right panels: quantification of LC3/STX17-positive cells in the presence or absence of BafA1 (one-way ANOVA; **P = 0.0060 vs. control; # P = 0.0136 vs. control BafA1) and quantification LC3/STX17-positive puncta per cells (n = 50 cells; one-way ANOVA; **P < 0.0001 vs. shTCHP BafA1).Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.

Fig 5: Effect of GA on autophagosome–lysosome fusion and degradation.LO2 cells were transfected with GFP-mCherry-LC3 for 24 h, and treated with TS (0.5 µl/ml) and/or GA (30 µM) for 24 h, the fluorescence images were captured by microscopy, scale bar = 30 µm (A). The lysosome and autophagosome markers Vamp 8 and Stx17 protein levels were detected by western blot and RT-PCR (B), their interactions were detected by co-immunoprecipitated (C) and effect of GA on lysosomal integrity were measured (D) in rat liver. C57bl/6 mice (n = 8) cotreated D/L with or without CQ (60 mg/kg) or GA (40 mg/kg) for 5 days (n = 8), and the expression of LC3-B, P62 and cleaved Parp1 (E), ALT and AST (F) were measured. Data are shown as mean ± SD, statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.