Fig 2: HIFAL is essential for the HIF-1a transcription activities.a The HRE element transcription activity is inhibited by HIFAL-LNA upon hypoxia. b–d HIFAL knockdown decreases the binding of HIF-1a to the promoter of LDHA (b) and PDK1 (c). HIFAL levels are shown in (d). e, f HIFAL knockout decreases the binding of p300 to the promoter of LDHA (e) and the histone H3K9 acetylation on the LDHA promoter (f). Primers spanning the LDHA locus [LDHA(A)], -873~-697bp[LDHA(B)] and +631~+823 bp[(LDHA(C)] were used. g HIFAL knockout decreases the chromatin sensitivity to micrococcal nuclease. h, The binding kinetics of HIF-1a to PKM2 and PHD3 keeps increasing after hypoxia in the presence of HIFAL. HIF-1a co-immunoprecipitation (Co-IP) assay was performed at indicated time points after hypoxia and normalized to the corresponding HIF-1a protein levels. i Distribution profiles of HIF-1a binding genes flanking the transcription start sites (TSS) in HIFAL WT or null MDA-MB-231 cells under hypoxia as determined by ChIP-seq. The 10 kb regions centered on all the TSSs of HIF-1a binding genes were shown. Red indicates enrichment, while blue indicates no enrichment. j, HIF-1a cannot bind to most of its target genes in the HIFAL null condition. k Binding motifs enriched within 1 kb of the promoter region. l A dual heatmap illustrating the co-occupancy peaks of HIF1a and HIFAL ChIP-seq in MBA-MD-231 cells. Peaks were rank from the highest amount of HIF-1a/HIFAL to the lowest, and were flanking with ±0.5 kb sequence of TSS. Red indicates enrichment, while blue indicates no enrichment. m The enrichment peaks of HIFAL and HIF-1a overlap significantly. Under the most stringent criteria, 2804 out of the 3054 (91.8%) HIFAL peaks are also bound by HIF-1a. n Binding motifs enriched within ±0.5 kb of the promoter region. HIF-1a and HIFAL shares the most frequent target sequence. o Representative HIF-1a target glycolysis gene loci are similarly enriched by both HIF-1a and HIFAL in HIFAL WT or null MDA-MB-231 cells. Graphs show means ± SD of experimental triplicates, p values were determined by two-sided unpaired t-test (a–f, h). Source data are provided as a Source Data file.

Fig 3: Correlation between the expression of LDHA and the level of immune infiltration in COAD. The correlation between the expression of LDHA and the immune infiltration of dendritic cells (A), CD 8+ cells (B), neutrophils (C), and macrophages (D) in COAD.

Fig 4: Hexokinase 2 and lactate dehydrogenase A protein expression in chronic hypoxia: Four MM cell lines and the bone marrow stromal cell line HS-5 were cultivated under hypoxic conditions and harvested at day 0, 1 and 7 for protein analysis. Representative Western blots for hexokinase 2 (HK2) (A) and lactate dehydrogenase A (LDHA) (C) are shown. Loading control: ß-tubulin. Densitometric quantifications of protein expression are shown in (B) for HK2 and (D) for LDHA. Signals were normalized to ß-tubulin expression. Bar graphs represent mean ±SEM of 3 independent experiments. One-way-ANOVA with Bonferroni’s post-hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Abbreviations: d = day; rel. = relative; kDa = kilodalton; HK2 = hexokinase 2; LDHA = lactate dehydrogenase A.

Fig 5: TKT promoted CRC cells metastasis through activating aerobic glycolysis.(A) Seahorse assays showed that TKT overexpression significantly promoted aerobic glycolysis. Left, representative curve, Right, quantification of three independent experiment of basal ECAR and maxi ECAR. (B–C) TKT promoted glucose consumption and lactate production in HCT116. (D) Seahorse assays showed that TKT knockdown significantly inhibited aerobic glycolysis, Left, representative curve, Right, quantification of three independent experiment of basal ECAR and maxi ECAR. (E–F) Relative glucose consumption and lactate production were detected in HCT116. (G–H) The content of F6P was detected in HCT116. (I) TKT promoted the expression of Glut1 and LDHA. (J–M) Wound-healing, migration and matrigel invasion assays were performed in HCT116 and SW480, *P < 0.05, **P < 0.01, ***P < 0.001.