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. 2021 Jul 16;10(7):52. doi: 10.1038/s41389-021-00338-7

Fig. 5. USP29/HIF1α axis in the regulation of glycolysis.

Fig. 5

a, b Huh7-IR/CR cells present increased acidification of cell medium. Cells were plated at the same numbers, and color changes of the culture medium were recorded 24 h after the seeding (a). pH values of the culture media were directly measured (b). n = 3 independent replicates. **P < 0.01; ***P < 0.001; Student’s t-test. c, d Sorafenib-resistant cells present with levels of glycolysis. Glucose uptake (c) and lactate production (d) were determined in Sorafenib-responsive Huh7 parental cells and in Sorafenib-resistant Huh7-IR and Huh7-CR cells. Normalized to cell numbers, Huh7-IR/CR cells showed higher glucose uptake and lactate production levels than Huh7 parental cells. n = 2 independent replicates. **P < 0.01; ***P < 0.001; Student’s t-test. e The mRNA levels of a selected subset of glycolysis-related genes were determined by quantitative RT-PCR in Sorafenib-responsive Huh7 parental cells and in Sorafenib-resistant Huh7-IR and Huh7-CR cells. High transcriptional levels of GLUT1, HK2, HK4, MCT3, and MCT4 were found in the Sorafenib-resistant cells. The expression of USP29 and HIF1α was unchanged between Huh7 parental cells and Huh7-IR/CR cells. n = 3 independent replicates. ns not significant; **P < 0.01; ***P < 0.001; Student’s t-test. f–i Depletion of USP29 or HIF1α diminishes the acidification of the culture medium in Sorafenib-resistant cells. Huh7-IR (f) and Huh7-CR (g) cells were plated at the same cell numbers 24 h after the transfection with siCtrl or ON-TARGET siRNAs against USP29 and HIF1α. Color changes were recorded 24 h later (f, g). pH values of the culture medium were directly measured in Huh7-IR (h) and Huh7-CR (i) cells. n = 2 independent replicates. *P < 0.05; Student’s t-test. j–m USP29 or HIF1α deficiency reduces glycolysis metabolism in Sorafenib-resistant cells. Huh7-IR (j, l) and Huh7-CR (k, m) cells were plated at the same cell numbers and transfected with siCtlr or ON-TARGET siRNAs against USP29 and HIF1α. Glucose uptake (j, k) and lactate production (l, m) were examined by determining relative luminescence (RLU) levels 24 h after siRNA transfection and normalized to cell numbers. n = 2 independent replicates. n, o Knockdown efficiencies of siRNAs against USP29 and HIF1α used in (j–m) as determined by immunoblotting. p, q Protein levels of hexokinase 2 (HK2), a major enzyme of the glycolytic pathway (p), and of hexokinase 4 (HK), the major liver hexokinase (q), were determined in a database of the whole proteomic analysis of needle biopsies from patients with HCC who responded to Sorafenib treatment (responder) or did not respond (non-responder). *P < 0.05; Student’s t-test.