Fig 2: ZBTB7A zinc finger mutant R399L is defective in binding to the glycolysis gene promoters and unable to repress glycolysis. (A, top) Domain architecture of ZBTB7A protein and the design of ZBTB7A zinc finger mutant R399L. Western blot analysis was performed to determine the expression of the R399L mutant and wild-type ZBTB7A. Both the R399L mutant and wild-type ZBTB7A were tagged with Xpress. (B) Xpress-tagged wild-type ZBTB7A (WT), R399L mutated ZBTB7A (R399L), and control Xpress vector were transfected in U2OS cells, and ChIP was performed with Xpress Ab. Primers specific to GLUT3, PFKP, or PKM promoter as in Figure 4A were used to determine DNA abundance with quantitative real-time PCR. Data shown are average values of three experiments, with error bars indicating mean ± SD. (C) Luciferase assays as described in Figure 4B were carried out to determine the ability of ZBTB7A R399L, with its wild-type counterpart as a control, to suppress the activity of GLUT3, PFKP, and PKM promoters, respectively. The data shown are average values of three experiments, with error bars indicating mean ± SD. (D) Plasmids encoding wild-type or R399L ZBTB7A were expressed in U2OS cells. Glucose consumption and lactate production were measured. Lactate produced and glucose consumed per 106 cells in a 1-h period of three experiments are shown. Error bars represent mean ± SD.

Fig 3: ZBTB7A suppresses glycolytic metabolism in mammalian cells. (A) U2OS cells were transfected with either siControl (Sicon) or three independent ZBTB7A targeting siRNA sequences (SiZBTB7A). (Bottom) The knockdown efficiency was determined by Western analysis. Culture media from an equal number of cells were collected 48 h after transfection for lactate measurement (top) and glucose consumption determination (middle). The numbers are mean ± SD from three independent experiments. (B) Zbtb7a-deleted MEFs (Zbtb7aΔ/Δ MEF) and control MEFs were similarly analyzed for lactate production (top) and glucose consumption (middle). The numbers represent mean ± SD from three independent experiments. (Bottom) Western blot analysis was performed to determine the level of ZBTB7A expression. (C, bottom) The expression of ZBTB7A was restored in Zbtb7a−/− MEFs. Zbtb7a+/+, Zbtb7a−/−, and Zbtb7a−/−+Zbtb7a MEFs were subjected to analysis of lactate production (top) and glucose consumption (middle). Data from three experiments are presented as mean values ± SD. (D) Zbtb7a-deleted and wild-type control MEFs were incubated with [1,2-13C]-glucose for 15 min prior to metabolite extraction and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The ratio of 13C-labeled to unlabeled (12C) metabolites was measured by LC-MS/MS and is presented as mean ± SD over four independent samples. Metabolites with P-values for pairwise comparisons are shown.

Fig 4: ZBTB7A inhibits proliferation and migration of Ishikawa cells. a The effect of plasmid pcDNA4-ZBTB7A overexpressed in Ishikawa cells. ZBTB7A inhibits growth (b), and migration (c) in Ishikawa cells

Fig 5: ZBTB7A gene mutation and protein interaction relationship analysis in UCEC. a ZBTB7A genes expression and mutation analysis in UCEC (cBioPortal). b protein–protein interactions of key genes. c Bar graph of enriched terms across input gene lists, colored by P-values. d Colored by cluster ID, where nodes that share the same cluster ID are typically close to each other. e Colored by P-value, where terms containing more genes tend to have a more significant P-value