Fig 1.
RNA-seq analysis reveals that high glucose treatment affects the function of HPCs widely.
(A) Volcano plot indicating the differently expressed genes identified by RNA-seq analysis in HPCs treated with High D-glucose (30 mM) and normal D-glucose (5.5 mM). (B) GO functional enrichment analysis was performed to analyze the functions of glucose-regulated genes. (C) KEGG enrichment analysis was performed to analyze the glucose-regulated pathways. (D) The mRNA levels of DJ-1 in were analyzed according to the RNA-seq profile.
Fig 2.
DJ-1 attenuates the high glucose-induced apoptosis of HPCs.
(A) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of DJ-1 in HPCs treated with mannitol (MG) or D-glucose were analyzed by Western blotting analysis and Image J software, respectively. (B) The mRNA levels of DJ-1 in HPCs treated with MG or D-glucose were analyzed by qRT-PCR analysis. (C) The cell viability was measured by CCK-8 assays in HPCs treated with MG, LG, HG and HG + DJ-1, respectively. (D) The cell viability was analyzed by EdU assays in HPCs treated with MG, LG, HG and HG + DJ-1, respectively. (E) The cell apoptosis was analyzed by Annexin V-FITC/PI apoptosis assay in HPCs treated with MG, LG, HG and HG + DJ-1, respectively. (F) The apoptotic rate of HPCs treated with MG, LG, HG and HG + DJ-1 was analyzed by GraphPad Prism 8 software. (G) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of cleaved caspase 3 and Bcl2 in HPCs treated with LG, HG and HG + DJ-1 were analyzed by Western blotting analysis and Image J software, respectively. MG: mannitol, LG: 5.5mM D-glucose, MG: 15mM D-glucose, HG: 30mM D-glucose, DJ-1: transfected with 1 μg/mL DJ-1-Flag-pcDNA3.1 plasmid. Data are shown as mean ± SD and representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3.
DJ-1 increases the expression of NF-κB and AP-1 by inducing the accumulation of ERK1/2 phosphorylation in HPCs.
(A) Custom VennMap diagrams were generated by using the RNA-seq data from HPCs treated with LG, MG and HG, respectively. (B) Heat map was performed to analyze the MAPK-regulated genes from the 110 changed genes of HPCs with the treatment of LG and HG. (C) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of HPCs transfected with siDJ-1#1, siDJ-1#2, siDJ-1#3 were examined by Western blotting analysis and Image J software, respectively. (D) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of ERK1/2 and p-ERK1/2 in HPCs transfected with siDJ-1#3 were analyzed by Western blotting analysis and Image J software, respectively. (E) and (F) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of ERK1/2 and p-ERK1/2 in HPCs treated with LG, HG and HG + DJ-1 were analyzed by Western blotting analysis and Image J software, respectively. (G) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of NF-κB p65 and AP-1 in HPCs treated with LG, HG and HG + DJ-1 were analyzed by Western blotting analysis and Image J software, respectively. MG: mannitol, LG: 5.5mM D-glucose, MG: 15mM D-glucose, HG: 30mM D-glucose, DJ-1: transfected with 1 μg/mL DJ-1-Flag-pcDNA3.1 plasmid. Data are shown as mean ± SD and representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 4.
Inhibition of ERK1/2 blocks the DJ-1-induced suppression of apoptosis in HPCs.
(A) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of NF-κB p65 and AP-1 in HPCs treated with LG, HG, HG + DJ-1 and HG + DJ-1 + temuterkib were analyzed by Western blotting analysis and Image J software, respectively. (B) Representative Western blot images and quantitative statistical bar graphs of protein expression levels of cleaved caspase 3 and Bcl2 in HPCs treated with LG, HG, HG + DJ-1 and HG + DJ-1 + temuterkib were analyzed by Western blotting analysis and Image J software, respectively. (C) The cell viability was measured by CCK-8 assays in HPCs treated with LG, HG, HG + DJ-1 and HG + DJ-1 + temuterkib, respectively. (D) The cell viability was analyzed by EdU assays in HPCs treated with LG, HG, HG + DJ-1 and HG + DJ-1 + temuterkib, respectively. (E) The cell apoptosis was analyzed by Annexin V-FITC/PI apoptosis assay in HPCs treated with LG, HG, HG + DJ-1 and HG + DJ-1 + temuterkib, respectively. (F) The apoptotic rate of HPCs treated with LG, HG, HG + DJ-1 and HG + DJ-1 + temuterkib were analyzed by GraphPad Prism 8 software. MG: mannitol, LG: 5.5mM D-glucose, MG: 15mM D-glucose, HG: 30mM D-glucose,temuterkib: 1μM, DJ-1: transfected with 1 μg/mL DJ-1-Flag-pcDNA3.1 plasmid. Data are shown as mean ± SD and representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.