Fig 2: High glucose (HG) effect on HIF-1a expression ((A)—protein, Western blot analysis; (C)—mRNA, RT-qPCR analysis), ChREBP expression ((B)—protein, Western blot analysis; (D)—mRNA, RT-qPCR analysis), expression of chosen HIF-1 target genes (E)—mRNA, RT-qPCR analysis) and the efficiency of ChREBP binding to the promoter region of HIF1A gene ((F)—end-point PCR analysis; (G)—relative fold enrichment of the target HIF1A gene over IgG sample) in RPTEC and D-RPTEC. Cells were cultured for 24 h under normoxic or under hypoxic (1% O2) conditions in the presence of normal (5.6 mM) or high (30 mM) glucose concentration. Then, 24.4 mM mannitol was added to the control media to ensure the same osmolarity. Chromatin was immunoprecipitated with anti-ChREBP antibody. PCR analyses were performed with specific primes for HIF1A gene, as described in 4.7. Values are means ± SEM for 3–5 experiments. Statistical significance: a p < 0.05 versus corresponding values in the presence of 5.6 mM glucose; b p < 0.05 versus corresponding values under normoxic conditions; c p < 0.05 versus corresponding values for control cells, i.e., RPTEC.

Fig 3: High glucose (HG) effect on HIF-1a expression ((A)—protein, Western blot analysis; (C)—mRNA, RT-qPCR analysis), ChREBP expression ((B)—protein, Western blot analysis; (D)—mRNA, RT-qPCR analysis), expression of chosen HIF-1 target genes (E)—mRNA, RT-qPCR analysis) and the efficiency of ChREBP binding to the promoter region of HIF1A gene (F)—end-point PCR analysis; (G)—relative fold enrichment of the target HIF1A gene over IgG sample) in HK-2 cells. Cells were cultured for 24 h under normoxic or under hypoxic (1% O2) conditions in the presence of normal (5.6 mM) or high (30 mM) glucose concentration. Then, 24.4 mM mannitol was added to the control media to ensure the same osmolarity. Chromatin was immunoprecipitated with anti-ChREBP, anti-RNA polymerase II (a positive control), and anti-IgG (a negative control) antibodies. PCR analyses were performed with specific primers for genes encoding GAPDH or HIF-1a, respectively, as described in 4.7. Values are means ± SEM for 3–5 experiments. Statistical significance: a p < 0.05 versus corresponding values in the presence of 5.6 mM glucose; b p < 0.05 versus corresponding values under normoxic conditions.