Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 2 Hypoxic induction of HIF pathway proteins in RCC cell lines. HIF pathway proteins were analysed by Western blotting following incubation of Caki-1, Caki-2 and 786-0 in decreasing oxygen tensions for 8 hrs. Bar charts show protein levels of HIF-1α (A, F, K), HIF-2α (B, G, L), GLUT1 (C, H, M), cyclin D1 (D, I, N) and BNIP3 (E, J, O) relative to a positive control (hypoxia-treated T24 cell lysate) which was loaded on each gel and referenced to β-actin. Data represent mean +SD from three independent experiments. Statistical significance was evaluated by One-way ANOVA with Dunnett’s Multiple Comparison Test; * p<0.05, ** p<0.01 *** p<0.001. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3; ND, not detected.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Incubation, Positive Control, Comparison

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 3 Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in Caki-1 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, HIF-2α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. Protein levels of HIF-1α and BNIP3 were decreased by ascorbate treatment under mild hypoxia (1–10% O2). Other proteins were not affected. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Control

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 5 Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in 786-0 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, HIF-2α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. There was no consistent difference of protein levels after ascorbate treatment under any condition. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Control

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 4 Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in Caki-2 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. Protein levels of HIF-1α were increased after ascorbate treatment at 1% and 0.1% O2. Expression of all HIF targets was unchanged. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Control, Expressing

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 7 Proline hydroxylation of HIF-1α in ccRCC cell lines with or without intracellular ascorbate. Western blot analysis confirmed expression of HIF-1α in the 786-HIF1 clones at P4 which decreased with increasing passage number (P5-P6). A hypoxia-treated T24 cell lysate was used as a positive control for HIF-1α (+) and β-actin as a loading control (A). Cell growth was monitored for four days, showing slower growth of 786-HIF1 clones compared to parental cells, increasing with repeated passaging. Cell numbers are per well of a 12-well plate. Data shown as mean +SD of three independent experiments. Statistical significance compared to parental 786-0 cells was assessed using 2-way ANOVA with Bonferroni post test (B). The effect of 500 μM ascorbate on P564 hydroxylation of HIF-1α was measured in parental 786-0 and 786-HIF1 cells, and in Caki-1 cells under ambient oxygen conditions or after hypoxia treatment (8 hrs at 1% O2) using Western blot. No hydroxy-HIF-1α was detected in parental 786-0 or Caki-1 cells (C). Densitometry analysis shows 1.5 fold increased hydroxylation in 786-HIF1 cells supplied with ascorbate compared to untreated 786-HIF1 cells (normalized to total HIF-1α content). Statistical significance was assessed by paired t-test (D). 786-HIF1 cells were treated with 50 μM–1 mM ascorbate and cell lysates subjected to Western blot for the detection of HIF-1α and hydroxy-HIF-1α (P564) (E). Relative hydroxy- HIF-1α (P564) levels (normalized to total HIF-1α levels) were increased after ascorbate treatment, as determined by densitometry analysis (F). Data shown as mean +SD from three independent experiments. Statistical significance compared to control condition was assessed by 1-way ANOVA with Bonferroni post test.* p<0.05, ** p<0.01, *** p<0.001. Abbreviations: HIF-1α, hypoxia-inducible factor-1α; P, passage number.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Expressing, Clone Assay, Positive Control, Control, Passaging

Journal: Cell & Bioscience

Article Title: VHL Ser65 mutations enhance HIF2α signaling and promote epithelial-mesenchymal transition of renal cancer cells

doi: 10.1186/s13578-022-00790-x

Figure Lengend Snippet: Gain of function of VHL mutations impact genome-wide HIF2α chromatin binding sites in 786-O cells. A , B Western blot determination of protein expression levels in 786-O cells stably expressing VHL WT and mutants, with or without treatment of the proteasome inhibitor MG132. C Genomic annotation of HIF2α ChIP-seq peaks from VHL WT mutant expressing cells. Genomic features of binding peaks were visualized in pie charts, where the demonstrated genomic features, including promoter regions within 1 kb, 1–2 kb and 2–3 kb; gene body (5′UTR, 3′UTR, exons, and introns); downstream elements, and distal intergenic regions. UTR, untranslated region. D Visualization of a HIF2α binding site at the promoter of a known hypoxia-regulated gene, VEGFA. E The top enriched motifs in the HIF2α ChIP-seq peaks determined by HOMER software. F , G Heatmap indication of HIF2α chromatin binding intensity based on ChIP-seq reads in 786-O cells expressing VHL WT vs. S65P mutant ( F ) or VHL WT vs. S65W mutant ( G ). Signals within 3 kb around ChIP-seq peak center are demonstrated in a descending order for each clustered HIF2α binding event (common or unique). Plots in the right panels of F or G display average signal of HIF2α binding at the indicated clustered regions

Article Snippet: Blocking buffer ((0.5% BSA in IP buffer (20 mM Tris–HCl, pH8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and cOmplete protease inhibitor cocktail (Roche)) pre-washed Dynabead protein G (Invitrogen) was incubated with antibody against HIF2α (AF2886, R&D Systems).

Techniques: Genome Wide, Binding Assay, Western Blot, Expressing, Stable Transfection, ChIP-sequencing, Mutagenesis, Software

Journal: Cell & Bioscience

Article Title: VHL Ser65 mutations enhance HIF2α signaling and promote epithelial-mesenchymal transition of renal cancer cells

doi: 10.1186/s13578-022-00790-x

Figure Lengend Snippet: Gain of function of VHL mutations enhance hypoxia signaling and glycolysis pathways. A , B Volcano plots of differentially expressed genes in 786-O cells expressing VHL mutations, S65P ( A ) or S65W ( B ), compared with VHL WT control. Significantly altered genes (adjusted p value < 0.05; fold change > ± 2) are indicated in green (upregulated) or dark blue (downregulated). Top ranked VHL mutation-influenced genes are selectively labeled. C , D GSEA-determined hypoxia ( C ) or glycolysis ( D ) pathways with S65W-influenced gene signature. Genes are ranked by their expression levels in 786-O cells expressing VHL mutation S65W. Plots in the right of C or D show HIF2α ChIP-seq signals of 5 kb around the transcriptional start sites (TSS) of altered genes in hypoxia ( C ) or glycolysis ( D ) pathways

Article Snippet: Blocking buffer ((0.5% BSA in IP buffer (20 mM Tris–HCl, pH8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and cOmplete protease inhibitor cocktail (Roche)) pre-washed Dynabead protein G (Invitrogen) was incubated with antibody against HIF2α (AF2886, R&D Systems).

Techniques: Expressing, Control, Mutagenesis, Labeling, ChIP-sequencing

Journal: Cell & Bioscience

Article Title: VHL Ser65 mutations enhance HIF2α signaling and promote epithelial-mesenchymal transition of renal cancer cells

doi: 10.1186/s13578-022-00790-x

Figure Lengend Snippet: Gain of function of VHL mutation promotes epithelial-mesenchymal transition (EMT). A , B GSEA-determined EMT pathways with VHL S65P ( A ) or S65W-influenced gene signature ( B ). Genes are ranked by their transcriptional levels in S65P ( A ) or S65W ( B ) expressed 786-O cells. Right panels of A or B indicate HIF2α ChIP-seq signals of 5 kb around TSS of EMT pathway genes influenced by VHL S65P ( A ) or VHL S65W ( B ). C Western blot of the EMT marker VIMENTIN protein levels across 786-O cell lines expressing empty vector (EV), VHL WT or mutants. D Left panel: representative images of invasion assays for control 786-O cells or the 786-O cell lines with VHL or VHL mutant expression. Right panel: quantitation of relative invasion for 786-O cells. E Left panel: representative images of migration assays for control 786-O cells or the 786-O cell lines expressing VHL or VHL mutants. Right panel: quantitation of relative migration for 786-O cells. In D,E, error bars s.d. n = 3 technical replicates. P values were assessed via the two-tailed Student’s t test

Article Snippet: Blocking buffer ((0.5% BSA in IP buffer (20 mM Tris–HCl, pH8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and cOmplete protease inhibitor cocktail (Roche)) pre-washed Dynabead protein G (Invitrogen) was incubated with antibody against HIF2α (AF2886, R&D Systems).

Techniques: Mutagenesis, ChIP-sequencing, Western Blot, Marker, Expressing, Plasmid Preparation, Control, Quantitation Assay, Migration, Two Tailed Test

Journal: Cell & Bioscience

Article Title: VHL Ser65 mutations enhance HIF2α signaling and promote epithelial-mesenchymal transition of renal cancer cells

doi: 10.1186/s13578-022-00790-x

Figure Lengend Snippet: VHL-S65P gene signature is associated with human kidney cancer progression. A Heatmap showing the expression profiles of genes differentially altered by VHL-S65P and directly bound by HIF2α. B ChIP-qPCR confirmation of HIF2α chromatin binding at the representative genes of VHL mutant signature in the cells expressing WT or mutant VHL (n = 3 replicates). C Analysis of mRNA expression of the VHL mutant signature genes in 786-O cells expressing VHL WT or mutants using quantitative RT-PCR (n = 3). D – E VHL-S65P signature is significantly upregulated in human renal cancers compared to normal kidney samples. F – I The expression of VHL-S65 signature positively correlate with EMT score ( F – H ) and hypoxia signaling score ( I )

Article Snippet: Blocking buffer ((0.5% BSA in IP buffer (20 mM Tris–HCl, pH8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and cOmplete protease inhibitor cocktail (Roche)) pre-washed Dynabead protein G (Invitrogen) was incubated with antibody against HIF2α (AF2886, R&D Systems).

Techniques: Expressing, ChIP-qPCR, Binding Assay, Mutagenesis, Quantitative RT-PCR

Journal: Cell & Bioscience

Article Title: VHL Ser65 mutations enhance HIF2α signaling and promote epithelial-mesenchymal transition of renal cancer cells

doi: 10.1186/s13578-022-00790-x

Figure Lengend Snippet: VHL-S65W gene signature is associated with human renal cancer severity and prognosis. A Heatmap showing the expression profiles of genes differentially influenced by VHL-S65W and directly occupied by HIF2α. B – D VHL-S65P signature is strikingly elevated in human kidney cancers compared to normal kidney specimens. E – H High expression of VHL-S65 signature positively correlate with elevated EMT ( E – G ) and hypoxia signaling score ( H ). I Kaplan–Meier graph showing a significant association between elevated expression of the VHL-S65W signature and shorter progression-free survival in a cohort of patients with renal cancers. J , K Kaplan–Meier graphs demonstrating significant associations between elevated VHL-S65W gene signature expression and reduced disease-specific survival ( J ) or disease-free survival ( K ). L , M Kaplan–Meier plots examining the risk of shortened overall survival in kidney cancer patients with higher expression of the VHL-S65W gene signature. In I – M , P values were calculated by a log-rank test

Article Snippet: Blocking buffer ((0.5% BSA in IP buffer (20 mM Tris–HCl, pH8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and cOmplete protease inhibitor cocktail (Roche)) pre-washed Dynabead protein G (Invitrogen) was incubated with antibody against HIF2α (AF2886, R&D Systems).

Techniques: Expressing