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: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: Sequences of the primers.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques:

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: The effect of TGF-β1 on cell transdifferentiation was analyzed by comparing the expression of COL1A1, HIF-1α, and α-SMA using TGF-β1-treated cells at different concentrations. ( A ) Western blot analysis of col1a1, HIF-1α, and α-SMA in cells of different groups. β-actin was used as loading control. ( B ) Quantitative analysis of COL1A1, HIF-1α, and α-SMA is shown in A. ( C ) Cells were treated with TGF-β1 at different concentrations, and mRNA was extracted from the treated groups. mRNA expression was determined by qPCR and normalized to β-actin. * p < 0.05 vs. 0 ng/mL group; # p < 0.05 vs. 2.5 ng/mL group; & p < 0.05 vs. 5 ng/mL group; % p < 0.05 vs. 7.5 ng/mL group. Results are expressed as means ± SD. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: The effect of TGF-β1 on cell transdifferentiation was analyzed by comparing the expression of COL1A1, HIF-1α, and α-SMA using TGF-β1-treated cells for different times. ( A ) Western blot analysis of COL1A1, HIF-1α, and α-SMA in cells of different groups. β-actin was used as a loading control. ( B ) Quantitative analysis of COL1A1, HIF-1α, and α-SMA is shown in A. ( C ) Cells were treated with TGF-β1 at different times, and mRNA was extracted from the treated groups. mRNA expression was determined by qPCR and normalized to β-actin. * p < 0.05 vs. 0 h group; # p < 0.05 vs. 2 h group; % p < 0.05 vs. 4 h group; $ p < 0.05 vs. 6 h group; ** < 0.05 vs. 12 h group. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: The effect of TGF-β1 on cell transdifferentiation was analyzed by comparing the expression of HIF-1α, SMAD 3, and PSMAD3 using TGF-β1-treated cells. ( A ) Western blot analysis of HIF-1α, SMAD 3, and PSMAD3 in cells of different groups. β-actin was used as a loading control. ( B ) Quantitative analysis of HIF-1α, P SMAD 3/ SMAD 3 is shown in A. * p < 0.05 vs. 0 h group; # p < 0.05 vs. 2 h group; % p < 0.05 vs. 4 h group. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: Cells were treated with DMSO, DMOG, and KC7F2 in the presence or absence of TGF-β1. mRNA and protein expression were examined to infer the degree of transdifferentiation. TGF-β1 was not present in the control group, the DMOG group, and the KC7F2 group. TGF-β1 was present in all of the TGF-β1 groups, the TGF-β1 + DMOG group, and the TGF-β1 + KC7F2 group. ( A ) Western blot analysis of COL1A1, HIF-1α, α-SMA, SMAD 3, and PSMAD3 in cells of different groups. β-actin was used as loading control. ( B ) Quantitative analysis of COL1A1, HIF-1α, P SMAD 3/ SMAD 3, and α-SMA is shown in A. ( C ) Different reagents were used to treat different groups of cells, and mRNA was extracted from groups. mRNA expression was determined by qPCR and normalized to β-actin. * p < 0.05 vs. control group; # p < 0.05 vs. DMOG group; $ p < 0.05 vs. TGF-β1 group. Results are expressed as means ± SD. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Expressing, Control, Western Blot

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: Cells were treated with NC-siRNA and HIF-1α-siRNA in the presence or absence of TGF-β1 and changes in mRNA and protein were examined to infer the degree of transdifferentiation. ( A ) Using flow cytometry, cell fluorescence intensity (transfection of siRNA that does not carry fluorescent groups) was measured, and 99.3% of the fluorescence intensity range was set as the threshold value (below which the cells were considered not to carry fluorescent groups), and transfection efficiency was calculated based on this threshold value. According to the threshold, cells exceeding the threshold were identified as successfully transfected with HIF-1α-siRNA (carrying fluorescent moieties). (HIF-1α-siRNA transfection efficiency was 71.9%) ( B ) Western blot analysis of COL1A1, HIF-1α,α-SMA, SMAD 3, and PSMAD3 in cells of different groups. β-actin was used as loading control. ( C ) Quantitative analysis of α-SMA, COL1A1, P SMAD 3/ SMAD 3, and HIF-1αis shown in B. ( D ) mRNA was extracted from the different groups, determined by qPCR, and normalized to β-actin. * p < 0.05 vs. NC-siRNA group; $ p < 0.05 vs. TGF-β1 + NC-siRNA group. Results are expressed as means ± SD. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Flow Cytometry, Fluorescence, Transfection, Western Blot, Control

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: Cells were treated with DMSO, DMOG, SIS3, and SIS3 + DMOG in the presence or absence of TGF-β1 and changes in mRNA and protein were examined to infer the degree of transdifferentiation. ( A ) Western blot analysis of different groups COL1A1, HIF-1α, SMAD 3, and PSMAD3. β-actin was used as loading control. ( B ) Quantitative analysis of COL1A1, HIF-1α, P SMAD 3/ SMAD 3, and α-SMA is shown in A. ( C ) Different reagents were used to treat different groups of cells, and mRNA was extracted. mRNA expression was determined by qPCR and normalized to β-actin. * p < 0.05 vs. control group; # p < 0.05 vs. TGF-β1; % p < 0.05 vs. DMOG; & p < 0.05 vs. TGF-β1 + DMOG group Results are expressed as means ± SD. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Western Blot, Control, Expressing

Journal: International Journal of Environmental Research and Public Health

Article Title: Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis

doi: 10.3390/ijerph19116775

Figure Lengend Snippet: A mouse PF model was established by nonexposure tracheal drip in C57BL/6N mice using saline or silica. The extent of PF in mice was inferred by Western blot, PCR, and IHC. Saline group (saline tracheal drip), silica group (silica particles tracheal drip), silica + NC group (silica particles tracheal drip, using saline intraperitoneal injection), and silica + KC7F2 group (silica particles, using KFC7F intraperitoneal injection). ( A ) Western blot analysis of COL1A1, HIF-1α,α-SMA, SMAD 3, and PSMAD3 in lung tissue of different groups. β-actin was used as loading control. ( B ) Quantitative analysis of COL1A1, HIF-1α, α-SMA P SMAD 3/ SMAD 3 is shown in A. ( C ) Quantitative analysis of the expression of mRNA. β-actin was used as an internal control. ( D ) H & E trichrome staining of mice lung tissue ( E ) Masson trichrome staining of mice lung tissue ( F ) IHC of COL1A1 protein. ( G ) IHC of HIF-1α protein. * p < 0.01 vs. saline group; # p < 0.01 vs. silica group. Results are expressed as means ± SD. ANOVA was used to compare multiple groups and pairwise comparisons were compared by LSD-t test. A value of p < 0.05 was considered to be statistically significant.

Article Snippet: For IHC staining, refer to the papers [ ]; 5 μm thick sections were incubated with rabbit anti-mouse primary antibodies against HIF-1α (1:200, 20960-1-AP, ProteinTech) and COL1A1 (1:1000, GB11022-3, Servicebio), followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG secondary antibody (1:8000, SA00001-2, Proteintech).

Techniques: Saline, Western Blot, Injection, Control, Expressing, Staining

Journal: Molecular Medicine Reports

Article Title: Suppression of CLEC3A inhibits osteosarcoma cell proliferation and promotes their chemosensitivity through the AKT1/mTOR/HIF1α signaling pathway

doi: 10.3892/mmr.2020.10986

Figure Lengend Snippet: Knockdown of CLEC3A suppresses the AKT1/mTOR/HIF1α pathway. (A) Scatter diagram demonstrating the co-expressing relationship between CLEC3A and AKT1 in mRNA level in patient OS tissues. (B) Expression levels of AKT1, mTOR, and HIF1α in the si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells was detected using western blotting. (C) Immunofluorescence was used to analyze the nuclear location of HIF1α in the si-NC- and si-CLEC3A-transfected MG63 cells (magnification, ×200). VEGF, GLUT1 and MCL1 mRNA expression levels were detected by reverse transcription-quantitative PCR in the si-NC- and si-CLEC3A-transfected (D) MG62 and (E) SaOS-2 cells. (F) VEGF, GLUT1, and MCL1 protein expression levels were detected in the si-NC- and si-CLEC3A-transfected cells by western blotting.*P<0.05. OS, osteosarcoma; CLEC3A, C-type lectin domain family 3 member A; NC, negative control; si, small interfering RNA; HIF1, hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor; GLUT1, glucose transporter 1; MCL1, induced myeloid leukemia cell differentiation protein Mcl-1.

Article Snippet: The cells were subsequently incubated with rabbit anti-HIF1α primary antibody (1:100; cat no. 20960-1-AP; ProteinTech Group, Inc.) overnight at 4°C.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA, Cell Differentiation

Journal: Molecular Medicine Reports

Article Title: Suppression of CLEC3A inhibits osteosarcoma cell proliferation and promotes their chemosensitivity through the AKT1/mTOR/HIF1α signaling pathway

doi: 10.3892/mmr.2020.10986

Figure Lengend Snippet: Restored AKT1/mTOR/HIF1α pathway expression reverses the effect of CLEC3A knockdown on biological functions. (A) Reverse transcription-quantitative PCR was used to detect the mRNA expression levels of VEGF, GLUT1 and MCL1 in si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells with or without treatment with SC79. (B) Cell Counting Kit-8 assays were used to detect the rate of cell proliferation in si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells with or without treatment with SC79. (C) Colony formation assays were used to detect the colony forming ability in si-NC- and si-CLEC3A-transfected cells with or without treatment with SC79 (diameter of dishes=6 cm). *P<0.05, **P<0.01. CLEC3A, C-type lectin domain family 3 member A; NC, negative control; si, small interfering RNA; VEGF, vascular endothelial growth factor; GLUT1, glucose transporter 1; MCL1, myeloid cell leukemia 1.

Article Snippet: The cells were subsequently incubated with rabbit anti-HIF1α primary antibody (1:100; cat no. 20960-1-AP; ProteinTech Group, Inc.) overnight at 4°C.

Techniques: Expressing, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Cell Counting, Negative Control, Small Interfering RNA