primary antibodies against hif1α  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: Morphological Features and HIF1-Dependent Processes in the Brain of Progeny of Female Rats Exposed to Maternal Hypoxia

doi: 10.3390/ijms27083421

Figure Lengend Snippet: Western blot analysis of HIF1α and GAPDH protein content in the fetal placenta (FP) and embryonic brain of Control and PMH groups at embryonic days e14, e16, e18, and e20. ( A ) Quantification of HIF1α protein content in the fetal placenta. Data were analyzed using an ordinary two-way ANOVA with Group (Control vs. PMH) and Time (e14, e16, e18, e20) as factors. The analysis revealed a significant Group × Time interaction (F(3,32) = 4.638, p = 0.0084) and a significant main effect of Time (F(3,32) = 4.638, p = 0.0084), while the main effect of Group was not significant. Sidak’s multiple comparisons test (Control vs. PMH within each embryonic day) showed significantly higher placental HIF1α protein content in the PMH group at e18 (adjusted p = 0.0014), with no significant differences at e14, e16, or e20. ** adjusted p < 0.01. Representative immunoblots are shown below the graph. ( B ) Quantification of GAPDH protein content in the fetal placenta. No significant differences between the Control and PMH groups were detected at any examined developmental stage. ( C ) Quantification of HIF1α protein content in the embryonic brain. No significant differences between the Control and PMH groups were detected at any examined developmental stage. Representative immunoblots are shown below the graph. ( D ) Quantification of GAPDH protein content in the embryonic brain. No significant differences between the Control and PMH groups were detected at any examined developmental stage. Representative immunoblots are shown below the graph. Bars represent mean ± SEM; dots indicate individual values, n = 5 per group.

Article Snippet: Membranes were blocked for 1 h in PBS containing 5% skimmed milk and incubated for 2 h at room temperature with rabbit primary antibodies against HIF1α (1:2000, AF1009, Affinity Biosciences, Cincinnati, OH, USA), LDHA (1:5000, AF7672, Affinity Biosciences, USA), G6PD (1:2000, DF6444, Affinity Biosciences, USA), GAPDH (1:5000, AF7021, Affinity Biosciences, USA), or β-Tubulin (1:5000, AF7011, Affinity Biosciences, USA).

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Morphological Features and HIF1-Dependent Processes in the Brain of Progeny of Female Rats Exposed to Maternal Hypoxia

doi: 10.3390/ijms27083421

Figure Lengend Snippet: qRT-PCR analysis of Hif1α and HIF1-dependent mRNA expression in adult brain structures of Control and PMH groups. ( A ) Relative Hif1α mRNA expression in the HPC, PFC, AMG, NAcc, VTA, and RN. PMH rats showed increased Hif1α mRNA expression in the HPC (Mann–Whitney U test, p = 0.0317) and decreased Hif1α mRNA expression in the VTA (unpaired Student’s t -test, p = 0.0336), while no significant differences were detected in the other analyzed structures. * adjusted p < 0.05 ( B ) Relative Gapdh mRNA expression in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( C ) Relative Hk1 mRNA expression in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( D ) Relative Ldha mRNA expression in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( E ) Relative G6pd mRNA expression in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. Bars represent mean ± SEM; dots indicate individual values, n = 5 per group.

Article Snippet: Membranes were blocked for 1 h in PBS containing 5% skimmed milk and incubated for 2 h at room temperature with rabbit primary antibodies against HIF1α (1:2000, AF1009, Affinity Biosciences, Cincinnati, OH, USA), LDHA (1:5000, AF7672, Affinity Biosciences, USA), G6PD (1:2000, DF6444, Affinity Biosciences, USA), GAPDH (1:5000, AF7021, Affinity Biosciences, USA), or β-Tubulin (1:5000, AF7011, Affinity Biosciences, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Morphological Features and HIF1-Dependent Processes in the Brain of Progeny of Female Rats Exposed to Maternal Hypoxia

doi: 10.3390/ijms27083421

Figure Lengend Snippet: Western blot analysis of HIF1-related protein content in adult brain structures of Control and PMH groups. ( A ) Quantification of HIF1α protein content in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( B ) Quantification of GAPDH protein content in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( C ) Quantification of LDHA protein content in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( D ) Quantification of G6PD protein content in the HPC, PFC, AMG, NAcc, VTA, and RN. No significant differences between the Control and PMH groups were detected in any analyzed brain region. ( E ) Representative immunoblots for the analyzed proteins in the corresponding brain structures. Bars represent mean ± SEM; dots indicate individual values, n = 5 per group.

Article Snippet: Membranes were blocked for 1 h in PBS containing 5% skimmed milk and incubated for 2 h at room temperature with rabbit primary antibodies against HIF1α (1:2000, AF1009, Affinity Biosciences, Cincinnati, OH, USA), LDHA (1:5000, AF7672, Affinity Biosciences, USA), G6PD (1:2000, DF6444, Affinity Biosciences, USA), GAPDH (1:5000, AF7021, Affinity Biosciences, USA), or β-Tubulin (1:5000, AF7011, Affinity Biosciences, USA).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: HIF1α-dependent induction of the T-Type calcium channel CaV3.2 mediates hypoxia-induced neuronal hyperexcitability

doi: 10.1101/2025.10.13.682038

Figure Lengend Snippet: NS20Y cells transfected with HIF1α show a significant upregulation of CaV3.2 in the (A) fluorescent reporter assay (Two-sided T Test, p = 0.013) (B) as well as in the dual Luciferase reporter assay (one-sample T Test, p = 0.037).

Article Snippet: Primary antibodies against HIF1α (Proteintech #20960-1-AP) were used in a concentration of 1:1000 in fish block over night at 4 °C.

Techniques: Transfection, Reporter Assay, Luciferase

Journal: bioRxiv

Article Title: HIF1α-dependent induction of the T-Type calcium channel CaV3.2 mediates hypoxia-induced neuronal hyperexcitability

doi: 10.1101/2025.10.13.682038

Figure Lengend Snippet: (A) primary neurons transduced with HIF1α show a significant upregulation of CaV3.2 in the Dual Luciferase reporter assay (one-sample T-Test, p = 0.016). (B) Primary neurons transduced with AAV-hSyn-HIF1α show a significantly increased WMFR compared to primary neurons transduced with a control virus (AAV-hSyn-GFP, two-way ANOVA HIF+/HIF– : F = 4.17, p = 0.049). (C) shows representative images of recordings of AAV-hSyn-GFP controls and AAV-hSyn-HIF1α with activity of every electrode shown in the left panel and bursting behavior on the right panel. (D) Artificial neural network analysis confirmed more hypoxia-like behavior of cells transduced with AAV-hSyn-HIF1α compared to control virus.

Article Snippet: Primary antibodies against HIF1α (Proteintech #20960-1-AP) were used in a concentration of 1:1000 in fish block over night at 4 °C.

Techniques: Transduction, Luciferase, Reporter Assay, Control, Virus, Activity Assay

Journal: bioRxiv

Article Title: HIF1α-dependent induction of the T-Type calcium channel CaV3.2 mediates hypoxia-induced neuronal hyperexcitability

doi: 10.1101/2025.10.13.682038

Figure Lengend Snippet: In murine OTCs (A) HIF1α shows a significant upregulation upon incubation in hypoxia in qPCR analysis compared to OTCs kept at normoxic conditions (two-sided paired T-Test, p = 0.05). (B) qPCR for Cacna1h revealed a drastic upregulation upon hypoxia compared to OTCs incubated in ambient oxygen concentration (two-sided paired T-Test, p = 0.009). (C) Human OTCs showed a significant upregulation of HIF1α upon exposure to hypoxia compared to OTCs kept at normoxic condition in western blot analysis (two-sided paired T-test, p = 0.04). Representative images of bands used for quantification are shown below (Ponceau S staining for WPN and HIF1α band at 120 kDa). (D) Analysis immunofluorescent stainings of CaV3.2 revealed a higher area fraction of CaV3.2 upon hypoxia compared to slices kept at normoxic conditions (two-sided paired T-Test, p = 0.03). (E) Representative images of immunofluorescent stainings of NeuN positive cells (neurons) and CaV3.2 staining. In normoxic conditions very little CaV3.2 expression is present whereas upregulation of CaV3.2 is visible on the apical dendrite of shown neuron. Scale bar corresponds to 10 µm.

Article Snippet: Primary antibodies against HIF1α (Proteintech #20960-1-AP) were used in a concentration of 1:1000 in fish block over night at 4 °C.

Techniques: Incubation, Concentration Assay, Western Blot, Staining, Expressing

Journal: bioRxiv

Article Title: HIF1α-dependent induction of the T-Type calcium channel CaV3.2 mediates hypoxia-induced neuronal hyperexcitability

doi: 10.1101/2025.10.13.682038

Figure Lengend Snippet: (A) Primary neurons exposed to the OGD/R model showed a significantly increased WMFR compared to neurons kept at normoxic conditions throughout the experiment (two-way ANOVA hypoxia × normoxia: F = 7.66, p = 0.009). (B) shows representative images of recordings of normoxic controls and hypoxia exposed OGD/R cells with activity of every electrode shown in the left panel and bursting behavior on the right panel. (C) Artificial neural network analysis confirmed similar behavior of hypoxia-exposed neurons to HIF+ (AAV-hSyn-HIF1α) cells, whereas neurons kept at normoxic conditions behaved rather like HIF-(AAV-hSyn-GFP) cells.

Article Snippet: Primary antibodies against HIF1α (Proteintech #20960-1-AP) were used in a concentration of 1:1000 in fish block over night at 4 °C.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Immunoblotting images showing the protein levels of HIF1α and its targets in 9 types of normal and cancer cells under normoxia (N) and hypoxia (H; 0.2% O 2 for 24 hours). n = 5-8. b, Confocal microscopy shows HIF1α expression in aerobic cell cultures of human PASMCs, PAECs, and AoECs. n = 3. c, HIF1α and α-smooth muscle actin (αSMA, a marker of VSMCs) expression in human lung tissues of transplantation-failed donors. n = 3. d, Glycolytic extracellular acidification rate (ECAR) in normoxic cell cultures determined by a Seahorse glycolytic stress assay. n = 5-9. e,f, PFKFB3 ( e ) and LDHA ( f ) protein levels in cells cultured in 21% or 0.2% O 2 for 24 hours. Fold change was calculated relative to corresponding normoxic cultures of each cell type. n = 5-8. g, Extracellular lactate levels in different normal and cancer cell types under normoxia or hypoxia (0.2% O 2 ) for 24 hours. n = 4-5. h, mRNA expression of HIF1α and its known transcriptional genes in aerobic cultures of PASMCs transfected with control siRNA (siCtrl) or HIF1α siRNA (siHIF1α). n = 9. i,j, Seahorse assays show ECAR ( i ) and oxygen consumption rate (OCR; j ) in PASMCs with HIF1α knockdown under normoxia. n = 3. k, Lactate secretion by PASMCs with HIF1α knockdown. Fold change was calculated relative to siCtrl-transfected cells. n = 7. All data were presented as mean ± SD. One-way ANOVA followed by Dunnett’s post-hoc test ( d ), Student’s t test or Mann-Whitney U test ( e-k ) was used when compared to aerobic culture of PASMCs or the matched cell type ( d-g ), or siCtrl-transfected PASMCs ( h-k ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Western Blot, Confocal Microscopy, Expressing, Marker, Transplantation Assay, Cell Culture, Transfection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Schematics shows medium conditioning and reapplication. b, Representative immunoblots of HIF1α and its regulatory proteins in PASMCs cultured in fresh growth medium (GM) or conditioned medium (CM). n = 3. c, Relative mRNA expression of HIF1α key target genes in glucose metabolism in PASMCs cultured in GM or CM. n = 7. d, Seahorse glycolytic stress test shows basal ECAR and glycolytic capacity in GM- or CM- cultured PASMCs. n = 5. e, Extracellular lactate levels in PASMCs grown in GM or CM. Fold change was calculated relative to GM-cultured cells. n = 10. f,g, Protein levels ( f ) and mRNA expression ( g ) of HIF1α and its transcriptional targets in PASMCs grown in GM or CM after transfection with control siRNA (siCtrl) or human HIF1α siRNA (siHIF1α). n = 3 ( f ) and 5 ( g ). h, LC-MS metabolomic profiling shows the levels of glycolytic metabolites G3P, pyruvate (PYR), and lactate (LAC) in PASMCs treated as described in panel f . n = 3. i, Seahorse glycolytic stress test shows basal ECAR and glycolytic capacity in CM-cultured PASMCs transfected with siCtrl or siHIF1α. n = 3. j, Extracellular lactate levels in GM or CM cultures of PASMCs with or without siHIF1α transfection. n = 5. All data are presented as mean ± SD. Student’s t test or Mann-Whitney U test ( c-e , i ), one-way ANOVA followed by Tukey’s post-hoc or Kruskal-Wallis test followed by Dunn’s post-hoc test ( g , h , j ) was used when compared to GM-cultured PASMCs ( c-e ), GM-cultured and siCtrl-transfected PASMCs ( g , h , j ), or siCtrl-transfected and CM-cultured PASMCs ( g-j ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Volcano plot of LC-MS metabolomic profiling shows the levels of 138 secreted metabolites in CM of PASMCs. n = 3. b, Quantitation of KIC/KMV (both have the same m/z ratio and are indistinguishable by LC-MS) and KIV in CM of PASMCs. ND: non-detectable. n = 3. c, Intracellular levels of KIC/KMV and KIV by LC-MS analysis in PASMCs cultured in GM or CM. Fold change was calculated relative to GM-cultured cells. n = 3. d, HPLC measurements of BCKAs in CM of PASMCs and basal GM. n = 3-6. e,f, Protein levels ( e ) and mRNA expression ( f ) of HIF1α and its target genes in PASMCs treated with KIC (100 μM), 50 μM of KMV or KIV, or all three BCKAs combined (KIC 100 μM, 50 μM KMV, and 50 μM KIV). n = 3 ( e ) and 5 ( f ). Fold change in f was calculated relative to vehicle control H 2 O treated cells. g, Protein levels of HIF1α, LDHA, and PDK1 in PASMCs cultured in GM or CM from PASMCs transfected with siRNAs for control (siCtrl), BCAT1 (siBCAT1), BCAT2 (siBCAT2 ) , or both BCAT1 and BCAT2 (siBCAT1/2). n = 3. h, HPLC determination of secreted BCKA levels in CM of PASMCs after transfection with siCtrl, siBCAT1, siBCAT2, or siBCAT1/2. n = 4. i-k, Representative immunoblotting images showing HIF1α and its target proteins PFKFB3 and LDHA in BCKA-treated human AoSMCs ( i ), CASMCs ( j ), and pericytes ( k ). n = 3. l, mRNA expression of HIF1α transcriptional genes in glycolysis in BCKA-treated human AoSMCs ( n = 4), CASMCs ( n = 5), and pericytes ( n = 6). Fold change was calculated relative to untreated control cells. All data are presented as mean ± SD. Student’s t test ( b-d ), one-way ANOVA followed by Dunnett’s test or Kruskal-Wallis test followed by Dunn’s test ( f , h , l) was applied when compared to GM ( b-d ), vehicle control H 2 O treated ( f , l ), or CM from siCtrl-transfected PASMCs ( h ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Cell Culture, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Reaction rates of KIC (250 μM), KMV (250 μM), KIV (250 μM), and all three BCKAs (200 μM of each BCKAs) determined by a competitive inhibition assay. Roxadustat (Roxa; 100 μM) was included as a positive control. n = 4. b, Inhibition curve and IC 50 value of three BCKAs for PHD2 hydroxylase activity. n = 4. c, The predicted docking sites and energy of KIC, KMV, and KIV on PHD2 enzyme (crystal structure accession ID: 2G19). d, Schematic diagram shows the metabolic fates of BCKAs. e, Representative immunoblots in untreated and BCKA-treated PASMCs. n = 3. f, 13 C 6 -KIC tracing of the labelled leucine (Leu) in PASMCs with BCAT1 , BCAT2 , or both ( BCAT1/2 ) silencing. n = 6. g, LC-MS measurements of metabolite levels in BCKA-treated PASMCs. Fold change was calculated relative to untreated control cells. n = 6. h,i, Intracellular L2HG levels ( h ) and protein levels of HIF1α, PFKFB3, and LDHA ( i ) in siCtrl or LDHA siRNA (siLDHA) transfected PASMCs with or without BCKA treatment. n = 4 ( h ) and 3 ( i ). j, L2HGDH mRNA expression in siCtrl or L2HGDH siRNA (siL2HGDH) transfected PASMCs with or without BCKA treatment. n = 4. k,l, Intracellular L2HG levels ( k ) and protein levels of HIF1α, PFKFB3, and LDHA ( l ) in siCtrl or siL2HGDH transfected PASMCs with or without BCKAs. n = 4 ( k ) and 3 ( l ). m, Immunoblotting shows protein levels in cell permeable trifluoromethylbenzyl (TFMB) ester of L2HG (500 μM) or D2HG (500 μM) treated PASMCs. n = 3. n, Immunoblots of HIF1α, PFKFB3, and LDHA in siCtrl or HIF1α siRNA (siHIF1α) transfected PASMCs with or without cell permeable L2HG (TFMB-L2HG) (500 μM). n = 3. o, Proposed mechanisms of BCKA-induced inhibition of PHD2 activity. All data are presented as mean ± SD. One-way ANOVA followed by Dunnett’s test ( a , f ) or Tukey’s test ( h , j , k ), Student’s t test or Mann-Whitney U test ( g ) was used when compared to no inhibitor control ( a ), untreated control cells ( g ), siCtrl-transfected and control or BCKA-treated PASMCs ( f , h , j , k ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Inhibition, Positive Control, Activity Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Transfection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Volcano plot from LC-MS metabolomic profiling showing the changes of 136 metabolites in PASMCs with or without BCKA treatment. n = 6. b, Top 10 Metabolite Set Enrichment Analysis (MSEA) pathways in BCKA-treated PASMCs. c, LC-MS measurements of glycolytic metabolites in BCKA-treated PASMCs. Fold change was calculated relative to untreated controls. n = 6. d, Extracellular lactate levels in BCKA-treated PASMCs. Fold change was calculated relative to untreated controls. n = 4. e,f, Protein levels ( e ) and mRNA expression ( f ) of HIF1α and its transcriptional targets in PASMCs transfected with siCtrl or siHIF1α followed by stimulation with BCKAs. Fold change in f was calculated relative to control cells. n = 3 ( e ) and 4 ( f ). g, LC-MS measurements show the levels of glycolytic metabolites in PASMCs treated as described in panel e . Fold change was calculated relative to siCtrl-transfected and untreated controls. n = 3. h, Seahorse glycolytic stress test demonstrates glycolytic capacity of PASMCs treated as described in panel e . n = 3. i, Extracellular lactate levels in PASMCs treated as described in panel e . Fold change was calculated relative to siCtrl-transfected and untreated controls. n = 5. All data are presented as mean ± SD. Student’s t test or Mann-Whitney U test ( c , d ), or one-way ANOVA followed by Tukey’s test ( f-i ) was conducted when compared to untreated controls ( c , d ), siCtrl-transfected and untreated or BCKA-treated PASMCs ( f-i ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Transfection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, mRNA expression of synthetic marker VIM and contractile marker ACTA2 in BCKAs (100 μM), TGFβ (2 ng/mL), or PDGF-BB (10 ng/mL) stimulated cells. Fold change was relative to untreated controls. n = 5. b, Representative immunoblots of synthetic marker proteins in BCKA-stimulated cells. n = 6. c, Confocal microscopy images and quantitation represent COL4 synthesis and deposition in BCKA-treated PASMCs. Fold change was relative to control cells. n = 5. d,e, Protein ( d ) and mRNA ( e ) expression of synthetic marker genes in PASMCs transfected with control siRNA (siCtrl) or human BCAT1 and BCKDHA1 siRNAs (siBCAT1+siBCKDHA1) with or without BCKA treatment. Fold change was calculated relative to siCtrl-transfected and untreated cells. n = 3 ( d ) and 6 ( e ). f,g, Protein ( f ) and mRNA ( g ) expression of synthetic marker genes in PASMCs transfected with siCtrl or human BCAT2 and BCKDK siRNAs (siBCAT2+siBCKDK) with or without BCKA treatment. Fold change was calculated relative to siCtrl-transfected and untreated cells. n = 3 ( f ) and 12 ( g ). h, COL1A1 and VIM protein expression in control and HIF1 α knockdown PASMCs in the presence or absence of BCKAs. n = 8. i, Confocal microscopy and quantitation showing COL1A1 synthesis and deposition in control and HIF1α knockdown PASMCs in the presence or absence of BCKAs. Fold change was relative to control cells. n = 6. j, Collagen gel contraction assay illustrating the relative contracted area of collagen gels after 8-hour of detachment in control and HIF1α knockdown PASMCs with or without BCKAs. n = 6. All data are presented as mean ± SD. One-way ANOVA followed by Dunnett’s ( a ) or Tukey’s post-hoc test ( e , g , i , j ), or Kruskal-Wallis test followed by Dunn’s test (COL4A1 in g ), or Mann-Whitney U test ( c ) was applied when compared to untreated controls ( a , c ), siCtrl-transfected and untreated or BCKA-treated PASMCs ( e , g , i , j ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Expressing, Marker, Western Blot, Confocal Microscopy, Quantitation Assay, Transfection, Collagen Gel Contraction Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a,b, mRNA ( a ) and protein ( b ) expression of key BCKA metabolic genes in human lung tissues of idiopathic PAH (IPAH) patients and transplantation-failed donors (Ctrl). Fold change was calculated relative to control donors. n = 10 individuals ( a ) and 8 individuals ( b ). c, The levels of BCKAs and BCAAs in the lungs of IPAH patients and control donors. Fold change was calculated relative to control donors. n = 7 individuals. d, Immunofluorescent images and quantitation of HIF1α protein in lung tissues of IPAH patients and control donors. Fold change was calculated relative to control donors. n = 5 individuals. e-g, Protein levels of four BCKA metabolic enzymes in lung tissues of control and PAH rats induced by MCT ( e ), hypoxia (10% O 2 , f ), or Sugen5416 and hypoxia treatments ( g ). n = 4 ( e , g ) and 3-4 ( f ) rats per condition. h, Immunofluorescence images and quantitation of HIF1α protein in control and MCT-treated rat lung tissues. n = 4 rats. All data are presented as mean ± SD. Student’s t test ( a , c , d ) or Mann-Whitney U test ( h ) was conducted when compared to lungs from failed donors ( a , c , d ) or control rats ( h ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Expressing, Transplantation Assay, Quantitation Assay, Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, mRNA expression of four BCKA metabolic enzymes, glycolytic gene PFKFB3 , and synthetic markers in PASMCs from IPAH patients and commercially available control donors. Fold change was relative to control donors. n = 5 individuals. b, Protein levels of four BCKA metabolic enzymes and COL4 in PASMCs from IPAH and commercially available control donors. Fold change was relative to control donors. n = 4-5 individuals. c, Volcano plot from LC-MS metabolomic profiling showing the levels of 139 metabolites in normal and IPAH PASMCs. n = 5 individuals. d, LC-MS measurements of KIC/KMV, BCAAs, and glycolytic metabolites in normal and IPAH PASMCs. Fold change was calculated relative to normal PASMCs. n = 5 individuals. e,f, mRNA expression of HIF1α transcriptional target genes in glucose metabolism ( e ) and protein expression of HIF1α, PFKFB3, and COL1A1 ( f ) in BCKA-treated IPAH-PASMCs. Fold change was calculated relative to untreated IPAH-PASMCs. n = 7 ( e ) and 8 ( f ) from 4 individuals. g, Lactate secretion by IPAH-PASMCs treated with or without BCKAs. Fold change was calculated relative to untreated IPAH-PASMCs. n = 8 from 4 individuals. h, Confocal microscopy and quantitation results showing collagen synthesis and deposition in IPAH-PASMCs treated with vehicle control or BCKAs. Fold change was calculated relative to untreated IPAH-PASMCs. n = 5 from 4 individuals. All data are presented as mean ± SD. Student’s t test or Mann-Whitney U test was used when compared to PASMCs from normal control donors ( a , b , d ) or untreated PASMCs from IPAH patients ( e-h ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Quantitation Assay, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Empagliflozin increases kidney weight due to increased cell size in the proximal tubule S3 segment and the collecting duct

doi: 10.3389/fphar.2023.1118358

Figure Lengend Snippet: Induction of Hif1α in intercalated cells by SGLT2 inhibition. (A) Overview images of Hif1α stained kidneys (brown) of untreated wildtype mice ( n = 4) and 8 weeks EMPA-treated mice (30 mg/kg/d; n = 8). (B) Quantification of total kidney Hif1α positive areas, total tubule tissue area and the resulting ratio using the ZEN Intellesis image segmentation software. (C) Immunofluores-cence costaining of Hif1α (black) with marker proteins for the proximal tubule (megalin; red), thick ascending limb (Tamm-Horsfall protein, THP, purple), distal convoluted tubule (calbindin; orange), principle (Aqp-2; yellow) and intercalated (V-ATPase; green) cells of the collecting duct. Hif1α was found in some calmodulin positive tubules but mostly in V-ATPase positive intercalated cells of cortical and medullary collecting ducts. Bar charts show mean values (±SEM) and asterisks indicate p < 0.05. EMPA, empagliflozin.

Article Snippet: Sections were incubated with the primary antibody against Hif1α (10006421, 1:10,000, Cayman Chemical, Ann Arbor, MI, United States) over night at 4 °C.

Techniques: Inhibition, Staining, Software, Marker