Fig. 8: HK1-dependent glycolysis regulates intestine enterocyte differentiation.

A, B Mouse SI organoids were infected with lentivirus expressing the HK1 shRNA or NTC shRNA and cultured in the presence of puromycin (2 μg/ml) to select for organoid cells with stable knockdown of HK1. The organoids with stable knockdown of HK1 were incubated for 5 d followed by extraction of cell lysates to measure IAP activity as an indication of differentiation (A). Total RNA was extracted, and qPCR performed; n = 3 biological repeats (B). C, D Mouse SI organoids were treated with 2-DG (5 mM) for 4 d followed by assessment of IAP activity (C). Expression of selective enterocyte markers was analyzed by qPCR; n = 3 biological repeats (D). E–H Human duodenum organoids were treated with 2-DG (5 mM) for 4 d and then analyzed for the expression of enterocyte markers (E) and the stem cell marker OLFM4 (F) by qPCR; n = 3 biological repeats. Representative image of organoid morphology following treatment with either 2-DG or control (G). Organoid numbers (n = 10 fields per group) (H, left panel)) and organoid size (n = 66 organoids per control group; n = 38 organoids per 2-DG treatment group) (H, right panel) were assessed. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.005. (I) Summary model illustrating the proposed role of BMP4/miR-181a-5p signaling pathway in intestinal cell proliferation and differentiation.