Fig 2: BRCA1 decreased activity of glycolysis.(A–C) Western blotting and quantification analysis of HK2 and IDH1 enzymes. (D) The pO2 was tested in the culture medium under the hypoxic condition for the indicated time. (E) Lactate release and glucose consumption were evaluated in cell culture media by enzymatic colorimetric assays. Values are represented as mean ± SD, n = 3 (**P < 0.01 and ***P < 0.001).

Fig 3: Elevated glucose destabilizes HIF-1a through increased PHD activity.(A) 293T WT cells in LG were treated with the indicated doses of PHD inhibitors CoCl2, daprodustat (Dap), and DMOG for 6 h to stabilize HIF-1a protein. (B) 293T WT cells in LG or HG were treated with 100 µM CoCl2 or 50 µM daprodustat and HIF-1a was assessed by Western blot. Relative changes in HIF-1a expression, normalized to ß-actin by densitometry, are shown below the blot. (C) HRE-luciferase plasmid was transfected into 293T WT cells exposed to acute or chronic glucose and/or treated with 100 µM CoCl2 for 24 h. Changes in HIF-1 transcriptional activity were measured via relative luminescence (normalized to transfection control) and analyzed by two-way ANOVA with Sidak’s multiple comparisons. (D) Metal-assisted protein quantification of HIF-1a and downstream target proteins from WT cells grown in LG or HG. Antibody metal labels: HIF-1a 161Dy, PDGFA 165Ho, VEGFA 164Dy, PHD3 151Eu, p-AKT 159Tb, and actin 171Yb. (E) qRT-PCR analysis of IDH1 mRNA in 293T WT cells grown in LG, 24 h HG, or chronic HG (n = 3, one-way ANOVA). (F) Corresponding IDH1 (47 kD) protein analysis. (G) ELISA quantification of 2-KG in 293T WT LG or HG cells. (H) Schematic showing the up-regulation of IDH1 and 2-KG production in HG, leading to PHD-mediated hydroxylation and degradation of HIF-1a.

Fig 4: SIL inhibit the activity of IDH1 by dephosphorylation. (A) The effects of SIL on the phosphorylation of IDH1 were analyzed using a Phos‐tag assay. (B, E) IDH1 proteins were purified using IDH1 antibody, and then, using pan‐phosphorylated antibody P‐(S‐T‐Y) and Phos‐tag assay to detect the phosphorylation level using western blot. (C, F) DNA sequencing example of R132H not mutation in both control (CON) and SIL treatment IDH1. (D, G) Enzymatic activity of IDH1 treated with control and different concentrations SIL (n = 5)

Fig 5: Downregulation of PIM2 inhibits the proliferation of MDS cell lines via the IDH1/HIF1A signaling pathway. (A) Cell proliferation was determined by the Cell Counting Kit-8 assay. Downregulation of PIM2 inhibited the proliferation of the SKM-1 cell line. The results are presented as the mean ± standard deviation of three replicates, and are representative of three independent experiments. *P<0.05 vs. control siRNA. Reverse transcription-quantitative polymerase chain reaction revealed that the expression of PIM2 is (B) negatively correlated with IDH1 and (C) positively correlated with HIF1A in patients with MDS. (D) Western blotting revealed that inhibition of PIM2 expression led to increased expression of IDH1 and decreased expression of HIF1A. MDS, myelodysplastic syndrome; PIM2, Pim-2 proto-oncogene, serine/threonine kinase; siRNA, small interfering RNA; IDH1, isocitrate dehydrogenase [NADP(+)]1, cytosolic; HIF1A, hypoxia inducible factor 1 subunit α.