Fig. 1
From: GOLM1 suppresses autophagy-mediated anti-tumor immunity in hepatocellular carcinoma
GOLM1 suppresses autophagy-mediated anti-tumor immunity in hepatocellular carcinoma. a, b Expression of GOLM1 immunohistochemical reaction in benign liver tissues (n = 48) and cancerous tissues (n = 161). Tan dyeing indicates positive GOLM1 staining in the cytoplasm of liver cells. c Kaplan-Meier cumulative survival curves of HCC patients grouped as low expression or high expression of GOLM1 (n = 101, cutoff value = 96.5). d Analysis of correlation between the expression of CD8 and GOLM1 in HCC tissues (n = 138). e Western blot by using an anti-mouse GOLM1 antibody showed successful gene knockout in H22 and MCA205 cells generated with a CRISPR vector carrying a scrambled guide RNA sequence. β-actin was used as a loading control. f, g Tumor growth curves of immunocompetent Balb/c mice subcutaneously inoculated H22 cells (f), and C57BL/6 mice inoculated MCA205 cells (g). Tumor progression is monitored 2-3 times per week and depicted as error bars of mean±SEM at each time point. Each group of tumor sizes contains 5 mice, and these results are representative of three independent experiments. h Golm1−/− H22 and control cells were implanted into in mouse liver to establish an orthotopic HCC model and implanted mice survival was observed. i, j At the time point of 10 days, MCA205 tumors in wild-type C57BL/6 mice were harvested and processed to detect the indicated cell populations by flow cytometry. Total tumor-infiltrating lymphocytes (TILs) percentage in CD45+ cell population (i), and in CD11b+ cell population (j) were shown. k, l At the time point of 10 days, MCA205 tumors in wild-type C57BL/6 mice were harvested and processed to detect the indicated proteins by immunofluorescence microscopy. Immunofluorescence staining images of CD8 and Cleaved-Caspase3 were represented (k) and the expression level was summarized in l (Scale bars: 100 μm). m, n MCA205 tumors in wild-type C57BL/6 mice were harvested and processed to detect IFNγ secretion by ELISpot assay. m Representative images of ELISpot responses from H22 (up) and MCA205 (down) tumors. Each well is represented a mouse tumor, and the quantitative data are shown in n. o Golm1+/+, Golm1+/−, and Golm1−/− H22 cells were treated with or without MTX for 24 h. Cell apoptosis was detected through staining with Annexin V plus vital dye DAPI followed by flow cytometry analysis and a quantitative summary is shown. p Golm1+/+ and Golm1−/− H22 cells were treated with or without MTX for 24 h. Quantification of ATP secretion from cell supernatants immediately collected and detected by chemiluminiscence assay. q Growth curves of immunocompetent mice bearing Golm1+/+, Golm1−/−, CD39 overexpressing Golm1+/+ and CD39 over-expressing Golm1−/− MCA205 tumors. r Golm1+/+ and Golm1−/− MCA205 cell lines stably expressing RFP-GFP-LC3 reporter protein were generated via lentivirus-mediated overexpression. The cells that exhibited a large number of RFP-GFP-LC3 dots after treatment of EBSS (3 h) were analyzed by confocal immunofluorescence. Quantification of cells with Red-GFP-LC3 puncta is shown. s Golm1+/+, Golm1+/−, and Golm1−/− H22 cells were treated with completed medium or Earle’s balanced salt solution (EBSS) for 3 h. Cell lysates were prepared to be available for western blot detection, the blots were exposed as indicated antibodies and further exposed to the respective secondary antibodies. Representative western blot analysis of autophagy-related proteins (left) and key components of AKT-mTOR signaling pathway (right). t WT, Golm1−/− and Golm1−/− Atg5−/− H22 cells were treated with or without EBSS for the indicated time. Quantification of ATP secretion from cell supernatants immediately collected and detected by chemiluminiscence assay. u Growth curves of immunocompetent mice bearing Golm1+/+, Golm1−/− and Golm1−/− Tsc2−/− MCA205 tumors. v Analysis of correlation between the expression of LC3 and GOLM1 in HCC tissues (n = 138, r = −0.3502, P = 0.0457). w, x Golm1+/+, Golm1+/−, and Golm1−/− H22 tumors in Balb/c mice were isolated at day 10 after implantation, and gene expression was analyzed by RNA sequencing (n = 3/group). Top functional pathway items by GO analysis and IPA (v). Heat map demonstrates type I interferon-related genes with a P value of less than 0.05 and a fold change of greater than 2 over the control group (w). y Tumor growth curves of Ifnar-−/− mice implanted with Golm1+/+ and Golm1−/− MCA205 cells. The quantitative variables between the two groups are analyzed by the Mann–Whitney U test (f, g, q, t, u, y) or unpaired Student’s t-test (b, i, j, l, n, o, p, r). Correlation between two groups is by the two-tailed Pearson’s correlation analysis (d, v). Quantitative data are represented as mean ± SEM; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001
