Journal: Frontiers in oncology

Article Title: DNA Polymerase Iota Promotes Esophageal Squamous Cell Carcinoma Proliferation Through Erk-OGT-Induced G6PD Overactivation.

doi: 10.3389/fonc.2021.706337

Figure Lengend Snippet: FIGURE 3 | Pol i activates G6PD through OGT-promoted O-GlcNAcylation. (A) The relative mRNA level of POLI and OGT in Pol i differentially expressed TE-1 and KYSE-150 cells. (B) the protein level of Pol i, OGT and G6PD. (C) O-GlcNAcylation of G6PD was detected after G6PD immunoprecipitation in ESCC cells. (D) the promoter of OGT (-2000 to +500 bp) was cloned into pGL-4 vector. The pGL4-OGT and internal control reporter vector pRL-TK were co-transfected into TE-1 and KYSE-150 cells. The relative OGT promoter activity was detected by dual-luciferase reporter assay. (E) Immunohistochemical staining of Pol i, OGT and protein O- GlcNAcylation in paraffin-embedded ESCC tissues. Scale bar = 100 mm. (F) level of OGT and protein O-GlcNAcylation in ESCC and adjacent tissues based on IHC score. (G) The correlation between Pol i and OGT, and the correlation between OGT and protein O-GlcNAcylation were evaluated based on IHC score in 114 tumor tissue samples. (H) Survival analysis based on the IHC score of protein O-GlcNAcylation in 114 ESCC samples. Kaplan–Meier survival analysis was applied. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against bactin (Beyotime Biotechnology), Pol i (Proteintech, Rosemont, IL, USA), G6PD (Abcam, Cambridge, MA, USA), OGT (Proteintech), O-GlcNAc (Invitrogen Life Technologies, Carlsbad, CA, USA), Erk and p-Erk (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C overnight.

Techniques: Immunoprecipitation, Clone Assay, Plasmid Preparation, Control, Transfection, Activity Assay, Luciferase, Reporter Assay, Immunohistochemical staining, Staining

Journal: Frontiers in oncology

Article Title: DNA Polymerase Iota Promotes Esophageal Squamous Cell Carcinoma Proliferation Through Erk-OGT-Induced G6PD Overactivation.

doi: 10.3389/fonc.2021.706337

Figure Lengend Snippet: FIGURE 6 | Pol i promotes ESCC cell proliferation through G6PD activation in vivo. (A) the tumor volume of xenograft nude mice with different Pol i expression. Mice were divided into three groups when the average tumor size grew up to 300 mm3. 5 mg/kg polydatin was intraperitoneally injected every other day. Same volume of normal saline was used as control. (B) relative G6PD activity in tumor tissue. (C) hematoxylin and eosin (HE) staining and immunohistochemical staining of Pol i, OGT and protein O-GlcNAcylation in tumors. **P < 0.01, ***P < 0.001. Scale bar = 100 mm.

Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against bactin (Beyotime Biotechnology), Pol i (Proteintech, Rosemont, IL, USA), G6PD (Abcam, Cambridge, MA, USA), OGT (Proteintech), O-GlcNAc (Invitrogen Life Technologies, Carlsbad, CA, USA), Erk and p-Erk (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C overnight.

Techniques: Activation Assay, In Vivo, Expressing, Injection, Saline, Control, Activity Assay, Staining, Immunohistochemical staining