Figures and data
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OGT is significantly upregulated in human and mouse tumor samples.
A-C) Boxplot showing mRNA expression level of Ogt, Normal and tumor samples (A), Individual stages (B), Nodal metastasis status (C). The plot was generated using the UALCAN online server. D-E) Boxplot showing protein expression level of OGT, Normal and tumor samples (A), Individual stages (B). The plot was generated using the UALCAN online server (https://ualcan.path.uab.edu/analysis.html). F) IHC analysis of OGT expression in normal colon tissues, primary colon tumor samples (from Human Protein Atlas, https://www.proteinatlas.org/), scale bar: 400μm. G) Western blot analysis of OGT and O-GlcNAc in normal, adjacent and tumor tissues in Apcminspontaneous tumor mice. H) HE staining and IHC staining of OGT in adjacent and tumor tissues in Apcmin spontaneous tumor mice, scale bar: left panel 275 μm, right panel 75μm, n=3 respectively. I) Schematic of AOM/DSS model of colitis-associated colorectal cancer (CAC). J) Western blot analysis of OGT and O-GlcNAc in normal, adjacent and tumor tissues in CAC model. K) HE staining and IHC staining of OGT in adjacent and tumor tissues in CAC model, scale bar: left panel 275 μm, right panel 75μm, n=4 respectively. human samples (A-F), mouse samples (G-K). Statistical significance was determined by Pearson test, unpaired Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Data represent the mean of ± SD.
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Epithelial OGT deletion inhibits mouse colorectal tumorigenesis.
A) Western blot analysis of OGT expression and tumor number in Ogt△IEC model. B) HE staining, scale bar: up panel 275 μm, bottom panel 20 μm, n=3 respectively. C) Real-time PCR analysis of mRNA expression in intestine. D) ELISA analysis in intestine. Statistical significance was determined by unpaired Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Data represent the mean of ±SD.
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OGT deficiency induces cGAS/STING-dependent the type I IFN pathway.
A-D) Real-time PCR analysis of mRNA expression in different Ogt knockout gRNA clones in MC38 (A), LLC (B), HT29 (C), B16-OVA (D) cells. E) Western blot analysis of protein expression in different Ogt−/− clones in MC38, LLC and B16-OVA cells. F) Western blot analysis of protein expression in Ogt−/− rescued MC38, LLC, HT29 and B16-OVA cells. G-H) Real-time PCR and western blot analysis of mRNA and protein expression in different Ogt−/−Mavs−/− double knockout clones in MC38 cells. I-K) Real-time PCR and ELISA analysis of expression in Ogt−/−cGAS−/− double knockout clones in MC38 (I), MC38 (J), HT29 (K) cells. L) Western blot analysis of protein expression in Ogt−/−cGAS−/− or Ogt−/−Sting−/− double knockout clones in MC38, HT29 and B16-OVA cells. M-N) BMDCs pre-treated with B16-OVA-Ogt−/− (L), B16-OVA-Ogt−/−cGAS−/− or B16-OVA-Ogt−/−Sting−/− double knockout (M) supernatant, and co-cultured with OT-1 T cell, then T cell proliferation was evaluated by flow cytometry, OVA257-264 as a positive control. Representative fluorescence-activated cell sorting histograms and statistical data are shown. Data are representative of two or three independent experiments. Statistical significance was determined by unpaired Student’s t-test, one-way ANOVA, two-way ANOVA, *p < 0.05,**p < 0.01,***p < 0.001, ns, no significant difference. Data represent the mean of ±SD.
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OGT deficiency causes DNA damage and accumulates cytosolic DNA.
A) The dsDNA in different Ogt−/− clones were determined by PicoGreen staining assay and extranuclear dsDNA was quantified by image J in MC38 cells. B) The dsDNA in different Ogt−/− clones were determined by anti-dsDNA fluorescence staining assay and extranuclear dsDNA was quantified by image J in MC38 cells. C) Western blot analysis of γH2AX and H2AX in different Ogt−/− clones in MC38, LLC and B16-OVA cells. D) The anti-γH2AX staining assay in different Ogt−/− clones and extranuclear dsDNA was quantified by image J. E) The DNA damage was determined by comet assay, and extranuclear dsDNA was analyzed by using CometScore. F) Western blot analysis of γH2AX and H2AX in Ogt−/− rescued clone in MC38, LLC, HT29 and B16-OVA cells. G) The DNA damage in rescued MC38 cells were determined by comet assay, and extranuclear dsDNA was analyzed by using CometScore. Data are representative of three or four independent experiments. Statistical significance was determined by unpaired Student’s t-test, one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Data represent the mean of ±SD.
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The C terminal of HCF-1 rescue DNA damage and the type I IFN pathway in Ogt−/− cells.
A) Volcano plot of proteins identified by LC–MS/MS in stably expressed exogenous OGT in OGT knockout HT29 cells. B) Immunoprecipitation and western blot of OGT knockout HT29 rescued cells. C) Immunoprecipitation and western blot of 293T cells. D) western blot of Ogt knockout MC38 rescued cells. E) Co-IP analysis of the interaction between OGT and different HCF-1 mutant. F) Real-time PCR analysis of mRNA expression in different HCF-1 isoforms in MC38 OGT knockout cells. G) Western blot analysis of protein expression in exogenous HCF-1C600 expression in MC38 Ogt knockout cells. H) The anti-dsDNA fluorescence staining in exogenous HCF-1C600 expression in MC38 OGT knockout cells. Data are representative of three or four independent experiments. Statistical significance was determined by one-way ANOVA, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Data represent the mean of ±SD.
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Ogt deficiency inhibits tumor progression through enhancing infiltration by CD8+ T cells.
A-B) Growth volume, weight and survival of Ogt+/+ or Ogt−/− MC38 tumors in C57BL/6J mice, n=5 respectively. C-D) Growth volume, weight and survival of Ogt+/+ or Ogt−/− LLC tumors in C57BL/6J mice, n=5 respectively. E-H) Flow cytometry analysis showing percentage of CD4+ and CD8+ T cells (E), IFN-γ+ (F), TNF-α+ (G), IFN-γ+TNF-α+ double positive (H) expressing intratumoral CD8+ T cells in MC38 tumors, subcutaneous tumor isolated at day 18 post-tumor inoculation, n=5 respectively. I) Growth volume and weight of Ogt+/+ or Ogt−/− MC38 in Rag2-/- mice, n=5 respectively. J-K) Growth volume, weight and survival of Ogt+/+ or Ogt−/− MC38 tumors injected with either control IgG or anti-CD8α at day 0, 7 and 14 post tumor inoculation in C57BL/6J mice, n=5 respectively. L-M) Ogt-/-rescued MC38 tumors in C57BL/6J mice, tumor growth volume and weight (L), survival (M). N-O) Ogt−/−cGAS−/− or Ogt−/−Sting−/− double knockout MC38 tumors in C57BL/6J mice, tumor growth volume and weight (N), survival (O). P) Flow cytometry analysis showing percentage of CD4+ and CD8+ T cells (N), CD8+ IFN-γ+ (O), CD8+ TNF-α+ (P) in Ogt−/−cGAS−/− or Ogt−/−Sting−/− double knockout MC38 tumors in C57BL/6J mice, subcutaneous tumor isolated at day 18 post-tumor inoculation. Q-R) Growth volume, weight and survival of Ogt+/+ or Ogt−/− MC38 tumors injected with either control IgG or anti-PD-L1 at day 7, 10 and 13 post tumor inoculation in C57BL/6J mice, n=5 respectively. S) Kaplan-Meier survival curves for colorectal cancer patients with low (n=207) or high (n=231) OGT transcripts in TCGA dataset. T) Progression-free survival curves for colorectal cancer patients with low (n=58) or high (n=58) OGT transcripts in TCGA dataset. U) Scatterplot presenting the association between the mRNA expression level of OGT and CD8+ T infiltration, Spearman’s r = −0.263, p = 9.75E-6, Spearman’s rank correlation test. Data are representative of two or three independent experiments. Statistical significance was determined by Spearman’s rank correlation test, unpaired Student’s t-test, one-way ANOVA, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Data represent the mean of ±SD.
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Combination therapy with OSMI-1 and anti-PD-L1 augmented T cells and antitumor immunity.
A) The anti-dsDNA fluorescence staining treated with 50 μM and 100 μM in MC38 cells, respectively. B-C) Western blot analysis of protein expression treated with 50 μM and 100 μM in MC38 and LLC cells, respectively. D) The anti-γH2AX fluorescence staining treated with 50 μM and 100 μM in MC38 cells, respectively. E-F) Growth volume, weight and survival of MC38 tumors injected with either control OSMI-1 or anti-PD-L1 in C57BL/6J mice. G-H) Growth volume, weight and survival of LLC tumors injected with either control OSMI-1 or anti-PD-L1 in C57BL/6J mice. I-K) Flow cytometry analysis showing percentage of CD4+ and CD8+ T cells (I), CD8+ IFN-γ+ cells (J), CD8+ TNF-α+ cells (K) in MC38 tumors, subcutaneous tumor isolated at day 18 post-tumor inoculation. L-N) Flow cytometry analysis showing percentage of CD4+ and CD8+ T cells (L), CD8+ IFN-γ+ cells (M), CD8+ TNF-α+ cells (N) in LLC tumors, subcutaneous tumor isolated at day 18 post-tumor inoculation. Data are representative of three or four independent experiments. Statistical significance was determined by unpaired Student’s t-test, one-way ANOVA, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Data represent the mean of ±SD.