hif2a  (Novus Biologicals)

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: A hypoxia‐like response mediated by HIF2A upregulates ANGPTL4 during senescence. (A) GSEA plots showing enrichment of the HYPOXIA gene set in 3 senescence models: RAS‐induced senescence in IMR90 (IMR90‐RAS) MEK‐induced senescence in human mammary epithelial cells (hMEC_MEK), and etoposide‐induced senescence in WI38 (WI38‐ETO). Normalized Enrichment Score (NES) and FDR q‐value are indicated. (B, C) MRC5 cells were infected with an empty vector (CTRL), or HIF1A (HIF1A OE) and HIF2A (HIF2A OE) expressing vectors. (B) Relative mRNA expression of HIF1A , HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA values are indicated. (C) Western blot analysis of HIF1A, HIF2A, ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments. (D) HIF2A peaks identified by Cut & Tag at the ANGPTL4 promoter in MRC5 cells infected with HIF2A (HIF2) or control (Babe) retroviral particles. Two independent experiments were performed (HIF2A‐Rep1, HIF2A‐Rep2). Both peaks contain a Hypoxia Response Element (ACGTG). (E) Western blot analysis of ANGPTL4 and the loading control GAPDH during RAF‐induced senescence (OIS). Representative picture of n = 3 independent experiments. (F) MRC5/RAF:ER cells were infected with lentiviral vectors encoding scramble (shSCR) or HIF2A shRNA (shHIF2A) and next treated (+) or not (−) with 4‐OHT to induce senescence (OIS). Relative mRNA expression of HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown. (G) ChIP‐qPCR assay to assess endogenous HIF2A binding on the ANGPTL4 promoter during OIS induced by RAF. Chromatin fractions derived from 4‐OHT‐treated and untreated MRC5/RAF:ER cells were subjected to immunoprecipitation with anti‐HIF2A antibody or IgG control. Primer sets were designed for regions 2179 bp (distal) and 369 bp (proximal) upstream of the ANGPTL4 TSS, and for Actin promoter as a control. Mean ± SEM of n = 3 independent experiments. Paired t ‐test values are indicated. (H) Western blot analysis of ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments.

Article Snippet: The following primary antibodies were used: ANGPTL4 (NBP2‐19016, Novus); tomato (AB0040‐200, Origene); HIF2A (Lau et al. ), p21 (M7202, Dako); γH2AX (2577S, Cell Signaling); CCL3 (2190–1, Proteintech); FURIN (18413–1‐AP Proteintech); and IL1A—Goat Polyclonal mouse‐IL1A (AF‐400‐NA, R&D systems).

Techniques: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Retroviral, shRNA, ChIP-qPCR, Binding Assay, Derivative Assay, Immunoprecipitation

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: ANGPTL4 promotes proinflammatory SASP and tumorigenesis in the lung. (A) Schematic representation of the experimental design of (B–D). LSL‐dTOM;Kras G12D mice were infected with a CRE‐encoding lentivirus. Lungs were retrieved and processed 15 to 20 weeks after (B), or 5 weeks post‐infection mice were treated with ANGPTL4 blocking antibody (bAb) for 10 weeks before analyses (C–D). (B) Lungs were prepared 15–20 weeks after CRE‐encoding lentivirus infection. Immunohistochemistry was performed against Tomato (to stain KrasG12D‐positive cells), ANGPTL4 and HIF2A. Representative picture of at least 10 lesions in 4 KT mice and 3 control mice. Scale bar: 100 μm. Quantifications of positive cells from 3 independent lungs are shown. (C) Neoplastic lesion quantification in mice treated with ANGPTL4 bAb (bAb) ( n = 9) or not treated (CTRL) ( n = 10). Left panel: Representative picture of hematoxylin–eosin staining; right panel: Quantification of neoplastic lesions. Mean ± SEM, unpaired t ‐test. (D) Immunohistochemistry analysis of IL1A staining in mouse neoplastic lung lesions treated with ANGPTL4 bAb (bAb), n = 17 lesions; and not treated (CTRL), n = 53 lesions. Left panel: Representative picture of the staining; scale bar: 100 μm. Right panel: Quantification result, percentage of positive cells per lesion. Mean ± SEM, Mann Whitney test. (E–G) GSEA plots showing enrichment of the “REACTOME_SENESCENCE_ASSOCIATED_PHENOTYPE_SASP”, “HALLMARK_INFLAMMATORY_RESPONSE” and “HARRIS_HYPOXIA” gene sets in LUAD tumors with high ANGPTL4 mRNA expression versus low ANGPTL4 mRNA expression.

Article Snippet: The following primary antibodies were used: ANGPTL4 (NBP2‐19016, Novus); tomato (AB0040‐200, Origene); HIF2A (Lau et al. ), p21 (M7202, Dako); γH2AX (2577S, Cell Signaling); CCL3 (2190–1, Proteintech); FURIN (18413–1‐AP Proteintech); and IL1A—Goat Polyclonal mouse‐IL1A (AF‐400‐NA, R&D systems).

Techniques: Infection, Blocking Assay, Immunohistochemistry, Staining, Control, MANN-WHITNEY, Expressing

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: A hypoxia‐like response mediated by HIF2A upregulates ANGPTL4 during senescence. (A) GSEA plots showing enrichment of the HYPOXIA gene set in 3 senescence models: RAS‐induced senescence in IMR90 (IMR90‐RAS) MEK‐induced senescence in human mammary epithelial cells (hMEC_MEK), and etoposide‐induced senescence in WI38 (WI38‐ETO). Normalized Enrichment Score (NES) and FDR q‐value are indicated. (B, C) MRC5 cells were infected with an empty vector (CTRL), or HIF1A (HIF1A OE) and HIF2A (HIF2A OE) expressing vectors. (B) Relative mRNA expression of HIF1A , HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA values are indicated. (C) Western blot analysis of HIF1A, HIF2A, ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments. (D) HIF2A peaks identified by Cut & Tag at the ANGPTL4 promoter in MRC5 cells infected with HIF2A (HIF2) or control (Babe) retroviral particles. Two independent experiments were performed (HIF2A‐Rep1, HIF2A‐Rep2). Both peaks contain a Hypoxia Response Element (ACGTG). (E) Western blot analysis of ANGPTL4 and the loading control GAPDH during RAF‐induced senescence (OIS). Representative picture of n = 3 independent experiments. (F) MRC5/RAF:ER cells were infected with lentiviral vectors encoding scramble (shSCR) or HIF2A shRNA (shHIF2A) and next treated (+) or not (−) with 4‐OHT to induce senescence (OIS). Relative mRNA expression of HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown. (G) ChIP‐qPCR assay to assess endogenous HIF2A binding on the ANGPTL4 promoter during OIS induced by RAF. Chromatin fractions derived from 4‐OHT‐treated and untreated MRC5/RAF:ER cells were subjected to immunoprecipitation with anti‐HIF2A antibody or IgG control. Primer sets were designed for regions 2179 bp (distal) and 369 bp (proximal) upstream of the ANGPTL4 TSS, and for Actin promoter as a control. Mean ± SEM of n = 3 independent experiments. Paired t ‐test values are indicated. (H) Western blot analysis of ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments.

Article Snippet: The membranes were then blocked for 1 h with 5% milk in TBS‐T 0.05% and subsequently incubated overnight at 4°C with primary antibodies in TBS‐T with 5% milk: α‐ANGPTL4 (sc‐373761, Santa Cruz, 1/250), α‐IL6 (sc‐28343, Santa Cruz, 1/500), α‐FURIN (18413‐1‐AP, Proteintech, 1/1000), α‐TUBULIN (T6199‐100, Sigma, 1/5000). α‐HIF1A (610958, BD Biosciences, 1/1000), HIF2A (NB100‐132, Novus Biologicals, 1/1000), INHBA (GTX108405, GeneTex 1/1000).

Techniques: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Retroviral, shRNA, ChIP-qPCR, Binding Assay, Derivative Assay, Immunoprecipitation

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: ANGPTL4 promotes proinflammatory SASP and tumorigenesis in the lung. (A) Schematic representation of the experimental design of (B–D). LSL‐dTOM;Kras G12D mice were infected with a CRE‐encoding lentivirus. Lungs were retrieved and processed 15 to 20 weeks after (B), or 5 weeks post‐infection mice were treated with ANGPTL4 blocking antibody (bAb) for 10 weeks before analyses (C–D). (B) Lungs were prepared 15–20 weeks after CRE‐encoding lentivirus infection. Immunohistochemistry was performed against Tomato (to stain KrasG12D‐positive cells), ANGPTL4 and HIF2A. Representative picture of at least 10 lesions in 4 KT mice and 3 control mice. Scale bar: 100 μm. Quantifications of positive cells from 3 independent lungs are shown. (C) Neoplastic lesion quantification in mice treated with ANGPTL4 bAb (bAb) ( n = 9) or not treated (CTRL) ( n = 10). Left panel: Representative picture of hematoxylin–eosin staining; right panel: Quantification of neoplastic lesions. Mean ± SEM, unpaired t ‐test. (D) Immunohistochemistry analysis of IL1A staining in mouse neoplastic lung lesions treated with ANGPTL4 bAb (bAb), n = 17 lesions; and not treated (CTRL), n = 53 lesions. Left panel: Representative picture of the staining; scale bar: 100 μm. Right panel: Quantification result, percentage of positive cells per lesion. Mean ± SEM, Mann Whitney test. (E–G) GSEA plots showing enrichment of the “REACTOME_SENESCENCE_ASSOCIATED_PHENOTYPE_SASP”, “HALLMARK_INFLAMMATORY_RESPONSE” and “HARRIS_HYPOXIA” gene sets in LUAD tumors with high ANGPTL4 mRNA expression versus low ANGPTL4 mRNA expression.

Article Snippet: The membranes were then blocked for 1 h with 5% milk in TBS‐T 0.05% and subsequently incubated overnight at 4°C with primary antibodies in TBS‐T with 5% milk: α‐ANGPTL4 (sc‐373761, Santa Cruz, 1/250), α‐IL6 (sc‐28343, Santa Cruz, 1/500), α‐FURIN (18413‐1‐AP, Proteintech, 1/1000), α‐TUBULIN (T6199‐100, Sigma, 1/5000). α‐HIF1A (610958, BD Biosciences, 1/1000), HIF2A (NB100‐132, Novus Biologicals, 1/1000), INHBA (GTX108405, GeneTex 1/1000).

Techniques: Infection, Blocking Assay, Immunohistochemistry, Staining, Control, MANN-WHITNEY, Expressing

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: A hypoxia‐like response mediated by HIF2A upregulates ANGPTL4 during senescence. (A) GSEA plots showing enrichment of the HYPOXIA gene set in 3 senescence models: RAS‐induced senescence in IMR90 (IMR90‐RAS) MEK‐induced senescence in human mammary epithelial cells (hMEC_MEK), and etoposide‐induced senescence in WI38 (WI38‐ETO). Normalized Enrichment Score (NES) and FDR q‐value are indicated. (B, C) MRC5 cells were infected with an empty vector (CTRL), or HIF1A (HIF1A OE) and HIF2A (HIF2A OE) expressing vectors. (B) Relative mRNA expression of HIF1A , HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA values are indicated. (C) Western blot analysis of HIF1A, HIF2A, ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments. (D) HIF2A peaks identified by Cut & Tag at the ANGPTL4 promoter in MRC5 cells infected with HIF2A (HIF2) or control (Babe) retroviral particles. Two independent experiments were performed (HIF2A‐Rep1, HIF2A‐Rep2). Both peaks contain a Hypoxia Response Element (ACGTG). (E) Western blot analysis of ANGPTL4 and the loading control GAPDH during RAF‐induced senescence (OIS). Representative picture of n = 3 independent experiments. (F) MRC5/RAF:ER cells were infected with lentiviral vectors encoding scramble (shSCR) or HIF2A shRNA (shHIF2A) and next treated (+) or not (−) with 4‐OHT to induce senescence (OIS). Relative mRNA expression of HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown. (G) ChIP‐qPCR assay to assess endogenous HIF2A binding on the ANGPTL4 promoter during OIS induced by RAF. Chromatin fractions derived from 4‐OHT‐treated and untreated MRC5/RAF:ER cells were subjected to immunoprecipitation with anti‐HIF2A antibody or IgG control. Primer sets were designed for regions 2179 bp (distal) and 369 bp (proximal) upstream of the ANGPTL4 TSS, and for Actin promoter as a control. Mean ± SEM of n = 3 independent experiments. Paired t ‐test values are indicated. (H) Western blot analysis of ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments.

Article Snippet: The following primary antibodies were used: ANGPTL4 (NBP2‐19016, Novus); tomato (AB0040‐200, Origene); HIF2A (Lau et al. ), p21 (M7202, Dako); γH2AX (2577S, Cell Signaling); CCL3 (2190–1, Proteintech); FURIN (18413–1‐AP Proteintech); and IL1A—Goat Polyclonal mouse‐IL1A (AF‐400‐NA, R&D systems).

Techniques: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Retroviral, shRNA, ChIP-qPCR, Binding Assay, Derivative Assay, Immunoprecipitation

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: ANGPTL4 promotes proinflammatory SASP and tumorigenesis in the lung. (A) Schematic representation of the experimental design of (B–D). LSL‐dTOM;Kras G12D mice were infected with a CRE‐encoding lentivirus. Lungs were retrieved and processed 15 to 20 weeks after (B), or 5 weeks post‐infection mice were treated with ANGPTL4 blocking antibody (bAb) for 10 weeks before analyses (C–D). (B) Lungs were prepared 15–20 weeks after CRE‐encoding lentivirus infection. Immunohistochemistry was performed against Tomato (to stain KrasG12D‐positive cells), ANGPTL4 and HIF2A. Representative picture of at least 10 lesions in 4 KT mice and 3 control mice. Scale bar: 100 μm. Quantifications of positive cells from 3 independent lungs are shown. (C) Neoplastic lesion quantification in mice treated with ANGPTL4 bAb (bAb) ( n = 9) or not treated (CTRL) ( n = 10). Left panel: Representative picture of hematoxylin–eosin staining; right panel: Quantification of neoplastic lesions. Mean ± SEM, unpaired t ‐test. (D) Immunohistochemistry analysis of IL1A staining in mouse neoplastic lung lesions treated with ANGPTL4 bAb (bAb), n = 17 lesions; and not treated (CTRL), n = 53 lesions. Left panel: Representative picture of the staining; scale bar: 100 μm. Right panel: Quantification result, percentage of positive cells per lesion. Mean ± SEM, Mann Whitney test. (E–G) GSEA plots showing enrichment of the “REACTOME_SENESCENCE_ASSOCIATED_PHENOTYPE_SASP”, “HALLMARK_INFLAMMATORY_RESPONSE” and “HARRIS_HYPOXIA” gene sets in LUAD tumors with high ANGPTL4 mRNA expression versus low ANGPTL4 mRNA expression.

Article Snippet: The following primary antibodies were used: ANGPTL4 (NBP2‐19016, Novus); tomato (AB0040‐200, Origene); HIF2A (Lau et al. ), p21 (M7202, Dako); γH2AX (2577S, Cell Signaling); CCL3 (2190–1, Proteintech); FURIN (18413–1‐AP Proteintech); and IL1A—Goat Polyclonal mouse‐IL1A (AF‐400‐NA, R&D systems).

Techniques: Infection, Blocking Assay, Immunohistochemistry, Staining, Control, MANN-WHITNEY, Expressing

Journal: JCI Insight

Article Title: Identification of HIF1A as a therapeutic target during SARS-CoV-2–associated lung injury

doi: 10.1172/jci.insight.191463

Figure Lengend Snippet: ( A ) Schematic diagram of vadadustat treatment in HIF reporter ODD-luc mice. ( B ) Representative ex vivo bioluminescence imaging of luciferase activity using IVIS imager at 2 hours after vadadustat i.p. injection. ( C ) Quantification of bioluminescence intensity at 2 hours and 4 hours after vadadustat i.p. injection. Data are presented as mean ± SD. ( D ) Schematic diagram of vadadustat treatment in C57BL/6 mice. ( E ) Hif1A or Hif2A immunoblotting was performed on protein isolated from whole lung tissue after treatment with vadadustat. Each column represents 1 animal. ( F and G ) Quantification of Hif1a and Hif2a protein after treatment with vadadustat for 3 days. Data are presented as mean ± SD. ( H ) Schematic diagram of WA1 infection (280 PFU) in K18-hACE2 mice treated with vadadustat. ( I ) Kaplan-Meier plots of K18-hACE2 mice with vehicle or vadadustat treatment. P values were calculated with the Mantel-Cox test. ( J ) Blinded histological injury scores of the lungs were quantified as described in the Methods. Data are represented as mean ± SEM. ( K ) Representative H&E staining images of lung tissue from vehicle- and vadadustat-treated mice. Scale bars: 50 μm. ( L ) Schematic diagram of MA10 infection (200 PFU) in BALB/c mice treated with vadadustat. ( M ) Kaplan-Meier plots of BALB/c mice with vehicle or vadadustat treatment. P values were calculated with the Mantel-Cox test. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( C ) or 2-tailed Student’s t test ( F , G , and J ).

Article Snippet: Membranes were probed with respective primary antibodies targeting Hif2a (NB 100-122, Novus; diluted 1:2000 in PBST), Hif1a (14179, Cell Signaling Technology; diluted 1:2000 in PBST), or α-tubulin (2144, Cell Signaling Technology; diluted 1:2000 in PBST) and incubated overnight at 4°C with gentle swirling.

Techniques: Ex Vivo, Imaging, Luciferase, Activity Assay, Injection, Western Blot, Isolation, Infection, Staining, Comparison

Journal: JCI Insight

Article Title: Identification of HIF1A as a therapeutic target during SARS-CoV-2–associated lung injury

doi: 10.1172/jci.insight.191463

Figure Lengend Snippet: ( A ) Mice were inoculated with 3 × 10 4 PFU of the murine-adapted SARS-CoV-2 strain (MA10) via oropharyngeal aspiration, and clinical outcomes were monitored over 7 days. ( B and C ) The survival rate in SARS-CoV-2–infected mice with whole-body deletion of Hif1a ( Hif1a fl/fl UBCCreER) or ( C ) Hif2a-deleted mice ( Hif2a fl/fl UBCCreER) compared to their respective Hif fl/fl litter mates. P values were obtained using the Mantel-Cox test. ( D ) Mice with a specific deletion of Hif1a in alveolar epithelial cells ( Hif1a fl/fl SPCCreER) and their Cre-inducible counterpart (SPCCreER) were infected with 3 × 10 3 PFU of the MA10 strain via oropharyngeal aspiration or mock infected, monitored for clinical outcomes and euthanized on day 4 to harvest BALF and lung tissue. ( E ) Albumin concentration in BALF was measured by ELISA. Data are represented as mean ± SEM. Two-tailed Student’s t test. ( F and G ) Viral load in BALF and lung tissue was detected by plaque assay. Gaussian distribution was assayed using the Shapiro-Wilk test. Unpaired 2-tailed Student’s t test or Mann-Whitney U test was applied to parametric or nonparametric data, respectively. ( H ) The lungs of infected SPCCreER and Hif1a fl/fl SPCCreER mice 4 days after infection were collected, fixed, and paraffin embedded. H&E staining was performed, and images were taken at ×10 magnification ( n = 5 or 8, respectively; representative images are shown). Scale bars: 200 μm. ( I ) The lung injury score was performed blindly. In the bar-and-whisker plots, the bounds of the boxes represent the 25%–75% interquartile range, the lines within the boxes represent the median, the whiskers represent data min/max, and there are no outlying values. Two-tailed Student’s t test. ( J ) Inflammatory molecules were measured using a multiplex array in the BALF from SPCCreER and Hif1a fl/fl SPCCreER SARS-CoV-2– or mock-infected mice. Volcano plot resulting from an unpaired 2-tailed Student’s t test with Welch’s correction comparing both groups. Molecules that were highly differentially secreted are emphasized in red. The column graphs represent individual results for IL-6, G-CSF, and IP-10 ( n = 10–12). Unpaired 2-tailed Student’s t tests with Welch’s correction or Mann-Whitney U test was applied to parametric or nonparametric data. Normality was established using the Shapiro-Wilk test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were probed with respective primary antibodies targeting Hif2a (NB 100-122, Novus; diluted 1:2000 in PBST), Hif1a (14179, Cell Signaling Technology; diluted 1:2000 in PBST), or α-tubulin (2144, Cell Signaling Technology; diluted 1:2000 in PBST) and incubated overnight at 4°C with gentle swirling.

Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Plaque Assay, MANN-WHITNEY, Staining, Whisker Assay, Multiplex Assay