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. 2023 Nov 8;78:101836. doi: 10.1016/j.molmet.2023.101836

Figure 5.

Figure 5

JAK2-dependent G6PD Y437 phosphorylation is required for IL-6 induced OSCC cell proliferation and tumor growth.

(A-B) We stably expressed G6PD shRNA in HSC-3 and CAL-27 cells, and then stably expressed WT Flag-G6PD or Flag-G6PD Y437F. The shRNA targeted the non-coding region of G6PD mRNA, and so that expression of WT Flag-G6PD and Flag-G6PD Y437F is resistant to G6PD shRNA. Cell proliferation rate was measured by BrdU incorporation assay (A) and colony formation assay (B) in the presence or absence of IL-6. The data are presented as the mean ± SD from 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

(C) HSC-3 cells were stably expressing G6PD shRNA, WT Flag-G6PD, Flag-G6PD Y437F or/and IL-6. Cells were subcutaneously injected in mice (n = 7). The images of two representative xenografts from each group were shown (left panel). The volumes of mice tumor xenografts were measured at indicated time points (right panel). ∗∗∗P < 0.001.

(D) Mice tumor xenografts (n = 7) were analyzed by immunohistochemical staining with an anti-Ki67 antibody. Scale bar, 80 μm.

(E) The specificity of the anti-G6PD pY437 antibody was examined by immunohistochemical staining using the IL-6 and WT Flag-G6PD-expressed mice tumor xenograft samples in the presence or absence of the indicated blocking peptides. The data are obtained from 3 independent experiments. Scale bar, 80 μm.

(F) Mice tumor xenografts (n = 7) were analyzed by immunohistochemical staining with the indicated antibodies. Scale bar, 100 μm.

(G-H) 50 human OSCC samples were analyzed by immunohistochemical staining with the indicated antibodies (G). The scores of immunohistochemical staining were analyzed by linear regression (H, 50 cases). The dots representing the samples that have the same scores for both IL-6 (Y-axis) and G6PD pY437 (X-axis) overlap each other, and are shown as only one dot in the graph. Scale bar, 100 μm.

(I) The scores of immunohistochemical staining were compared between stage I/II group (29 cases in total) and stage III/IV group (21 cases in total).

(J) The scores of immunohistochemical staining were compared between well differentiated group (22 cases) and moderately/poorly differentiated group (28 cases in total).