Fig. 4.

UTI improved the aerobic glycolysis induced by TGF-β1 in HK-2 cells. (A) The expression of COL1A1 I and ACTA2 was measured by qPCR (**P < 0.01, ****P < 0.0001). (B) The levels of Collagen I and α-SMA were detected by Western blot. (C) The grey value results of Collagen I and α-SMA were quantified. Tubulin was used as the loading control (*P < 0.05, **P < 0.01). (Dand E) The level of α-SMA was evaluated by immunofluorescence staining, the positive areas were quantified (Original magnification: × 200, scale: 50 μm, ***P < 0.001, ****P < 0.0001). (F) The expression of PKM2 and LDHA was detected by qPCR (ns for P > 0.05, **P < 0.01, ***P < 0.001). (Gand H) The levels of PKM2 and LDHA were detected by Western blot, and the grey value results were quantified. Tubulin was used as the loading control (ns for P > 0.05, *P < 0.05, **P < 0.01). (I) The levels of lactate, glucose and ATP in each group (ns for P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (J and K) The ECAR of glycolytic analysis and the OCR of mitochondrial stress analysis were executed out in HK-2 cells that received TGF-β1 and/or UTI, and the glycolysis rate, glycolytic capacity, basal respiration and maximal respiration were measured (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (L) The expression of AMPK and HIF-1α was measured by qPCR (*P < 0.05, **P < 0.01, ****P < 0.0001). (M and N) The levels of p-AMPK, AMPK and HIF-1α were detected by Western blot, and the grey value results were quantified. AMPK and Tubulin were used as the loading controls (*P < 0.05, **P < 0.01). Results were presented as mean ± SD of three individual experiments. The full-length blots/gels are presented in Supplementary Figs. 4–6.