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. 2019 Dec;33(12):13695–13709. doi: 10.1096/fj.201901353R

Figure 3.

Figure 3

PlGF inhibition up-regulates the PPP revealed by RNA-seq analysis and prevents G6PD activity impairment caused by high glucose in HRECs. After the confluent HRECs were treated with PBS control and PlGF antibody for 4 d, the cells were isolated for RNA isolation, sequencing, and bioinformatics analyses. Six replicates for each condition (control vs. antibody) were used for experimentation. The HRECs that were treated with l-glucose (25 mM), d-glucose (25 mM), and the PlGF antibody were collected for the G6PD enzyme activity assay. The assay experiments were repeated 3 times. A) Volcano plot represented by significantly up-regulated and down-regulated genes on the basis of the logFC and log10 [false discovery rate (FDR)]. Note that G6PD, PRDX1, PRDX3, and PRDX6 were marked in the plot. B) DEGs were classified in accordance with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The PPP, TGF-β, and other metabolic pathways were identified. C) Significantly up-regulated and down-regulated PPP genes in the HRECs treated with PBS control and PlGF antibody represented as a heat map (red color shows up-regulated genes, and green color shows down-regulated genes). D) PPP genes highlighted in red color (up-regulated) and green color (down-regulated) and represented in KEGG pathway. The DEGs, such as 1.1.1.49 (G6PD), 1.1.1363 (G6PD), 5.3.1.9 [glucose-6-phosphate isomerase (GPI)], 2.7.1.11 [phosphofructokinase liver/muscle type (PFKL/PFKM)], 4.1.2.13 [aldolase, fructose-bisphosphate A/C (ALDOA/ALDOC)], 4.1.2.4 [deoxyribose-phosphate aldolase (DERA)], 3.1.1.31 [6-phosphogluconolactonase (PGLS)], 5.1.3.1 [ribulose-phosphate 3-epimerase (RPE)], 2.7.2.1 [phosphoribosylpyrophosphate synthetase 1 (PRPS1)], and 2.7.1.165 [glycerate kinase (GLYCTK)], are involved in the PPP. E) WB results of G6PD, TKT, and TALDO in HRECs: β-actin was used as the protein loading control. F) Densitometry analysis of protein bands. O.D., optical density. G) G6PD enzyme activity under high glucose (HG; 25 mM d-glucose) and normal glucose (NG; 25 mM l-glucose for osmotic control) for 1, 2, and 3 d. H) G6PD enzyme activity of HRECs treated with NG control, HG, and HG + PlGF antibodies. The results were averaged from 3 independent experiments and expressed as a percentage relative to the NG condition. N.s., not significant. *P < 0.05, **P < 0.01.