hif2a  (R&D Systems)

Journal: International Journal of Cancer

Article Title: HIF2A as a prognostic and clinical therapeutic target in ovarian clear cell carcinoma

doi: 10.1002/ijc.70341

Figure Lengend Snippet: Nuclear expression of HIF2A is a prognostic factor in ovarian clear cell carcinoma. (A) HIF2A expression between clear cell carcinoma clinical samples ( n = 10) and normal counterparts ( n = 10) from the GSE29450 datasets ( n = 20). (B) HIF2A expression between clear cell carcinoma clinical samples ( n = 17) and nonclear cell carcinoma clinical samples ( n = 47) from our datasets ( GSE39204 ). (C) HIF2A expression between clear cell carcinoma clinical samples ( n = 25) and nonclear cell carcinoma clinical samples ( n = 30) from GSE65986 . (D) HIF2A expression between clear cell carcinoma cell lines ( n = 14) and nonclear cell carcinoma cell lines ( n = 24) from our datasets ( GSE29175 ). (E) Representative images of immunostaining for HIF2A nuclear expression. (F) Kaplan–Meier survival curves comparing progression‐free survival between patients with ovarian clear cell carcinoma with high ( n = 42) and low ( n = 22) HIF2A expression treated in Kyoto University Hospital. Log‐rank test indicates a significantly worse prognosis in the high‐expression group ( p = .003). (G) Kaplan–Meier survival curves comparing overall survival between patients with ovarian clear cell carcinoma with high ( n = 42) and low ( n = 22) HIF2A expression treated in Kyoto University Hospital. Log‐rank test indicates a significantly worse prognosis in the high‐expression group ( p = .005). (H) Kaplan–Meier survival curves showing poorer prognosis with high ssGSEA scores of the PID HIF2A PATHWAY gene set of MSigDB in the Washington University dataset (Kelly L Bolton et al. Clin Cancer Res. 2022) with overall survival times. The log‐rank test indicated a significantly worse prognosis in the high‐expression group ( p = 0.024). Data are presented as mean ± standard error of the mean.

Article Snippet: After blocking, the membranes were incubated overnight at 4°C with primary antibodies against HIF2A (D9E3, #7096, 1:1000 dilution, Cell Signaling Technology [CST], RRID: AB_10898028) and ACTB (#4967, CST, RRID: AB_330288 1:1000 dilution, Cell Signaling), followed by HRP‐conjugated secondary antibody (#7074, CST, RRID: AB_2099233,1:3000 dilution).

Techniques: Expressing, Immunostaining

Journal: International Journal of Cancer

Article Title: HIF2A as a prognostic and clinical therapeutic target in ovarian clear cell carcinoma

doi: 10.1002/ijc.70341

Figure Lengend Snippet: HIF2A knockdown enhances ROS production and mitochondrial function and suppresses tumor growth. (A)–(D) mRNA expression levels of HIF2A downstream genes were significantly downregulated in RMG1 cells following HIF2A knockdown under hypoxic conditions (1.5% O 2 ) for 24 h as determined by RT‐PCR. VEGFA (A), NDRG1 (B), GLUT1 (C), and IGFBP3 (D) expressions were normalized by ACTB. (E) Oxygen consumption rate (OCR) measurements in RMG1 cells (control, shHIF2A‐1, and shHIF2A‐2) under hypoxic conditions (1.5% O₂) for 24 h. Downward arrows indicate the injection points of oligomycin (1 μmol/L), FCCP (2 μmol/L), and rotenone/antimycin A (1 μmol/L each), from left to right. (F) Quantification of spare respiration in shcontrol and shHIF2A cell lines under 1.5% O 2 for 24 h. (G) Quantification of ATP production in shcontrol and shHIF2A cell lines under 1.5% O 2 for 24 h. (H) Quantification of maximal respiration in shcontrol and shHIF2A cell lines under 1.5% O 2 for 24 h. Quantification of mitochondrial ROS levels as mean fluorescence intensity (MFI) normalized to Hoechst staining in RMG1 cells under hypoxic conditions (1.5% O 2 ) for 24 h. (J) Representative fluorescence microscopy images of MitoSOX staining in shcontrol and shHIF2A cells, indicating the mitochondrial ROS levels. (K)–(N) HIF2A knockdown significantly inhibited tumor growth in RMG1 mouse xenograft models. Tumor growth curves comparing the shcontrol and shHIF2A groups (shHIF2A‐RMG1‐1 and shHIF2A‐RMG1‐2) are presented. Asterisks above the data points denote significance for shHIF2A‐1 and those below denote significance for shHIF2A‐2 (K). Tumor images from the shcontrol group (L). Tumor images from the shHIF2A‐1 group (M). Tumor images from the shHIF2A‐2 group (N). RMG1 cells transfected with either shcontrol or shHIF2A constructs were intradermally inoculated into mice ( n = 12 per group). Data are presented as mean ± standard error of the mean. RT‐qPCR data are presented as mean ± standard error of the mean from three independent experiments. * p <.05, ** p <.01, ** p <.001.

Article Snippet: After blocking, the membranes were incubated overnight at 4°C with primary antibodies against HIF2A (D9E3, #7096, 1:1000 dilution, Cell Signaling Technology [CST], RRID: AB_10898028) and ACTB (#4967, CST, RRID: AB_330288 1:1000 dilution, Cell Signaling), followed by HRP‐conjugated secondary antibody (#7074, CST, RRID: AB_2099233,1:3000 dilution).

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Injection, Fluorescence, Staining, Microscopy, Transfection, Construct, Quantitative RT-PCR

Journal: International Journal of Cancer

Article Title: HIF2A as a prognostic and clinical therapeutic target in ovarian clear cell carcinoma

doi: 10.1002/ijc.70341

Figure Lengend Snippet: NKT2152 enhances ROS production and mitochondrial function and induces an antitumor effect in CDX mouse models. (A)–(D) mRNA expression of HIF2A downstream genes was downregulated after treatment with 1 μM NKT2152 under 1.5% O 2 for 24 h in the RMG1 cell line as determined by RT‐PCR. VEGFA (A), NDRG1 (B), GLUT1 (C), and IGFBP3 (D) expressions were normalized by ACTB . (E)–(H) mRNA expression of HIF2A downstream genes was downregulated after treatment with 1 μM NKT2152 under 1.5% O 2 for 24 h in the KOC7C cell line as determined by RT‐PCR. VEGFA (E), NDRG1 (F), GLUT1 (G), and IGFBP3 (H) expressions were normalized by ACTB . (I) Measurement of OCR (oxygen consumption rate) in the RMG1 cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h (control, 1 μM, and 5 μM), as normalized by the cell protein concentration. Downward arrows show the injection points for oligomycin (1 μmol/L), FCCP (2 μmol/L), and rotenone/actinomycin (both 1 μmol/L) from left to right. (J) Quantification of spare respiration in the RMG1 cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h. (K) Quantification of ATP production in the RMG1 cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h. (L) Quantification of maximal respiration in the RMG1 cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h. (M) Measurement of OCR (oxygen consumption rate) in the KOC7C cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h (control, 1 μM, and 5 μM), as normalized by the cell protein concentration. Downward arrows show the injection points for oligomycin (1 μmol/L), FCCP (2 μmol/L), and rotenone/actinomycin (both 1 μmol/L) from left to right. (N) Quantification of spare respiration in the KOC7C cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h. (O) Quantification of ATP production in the KOC7C cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h. (P) Quantification of maximal respiration in the KOC7C cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h. (Q) Mean fluorescence intensity (MFI) of mitochondrial ROS, normalized to the MFI of Hoechst in the RMG1 cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h (control, 1 μM, and 5 μM). (R) Representative fluorescence images of MitoSOX in the RMG1 cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h (control, 1 μM, and 5 μM), demonstrating mitochondrial ROS levels. (S) Mean fluorescence intensity (MFI) of mitochondrial ROS, normalized to the MFI of Hoechst in the KOC7C cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h (control, 1 μM, and 5 μM). Representative fluorescence images of MitoSOX in the KOC7C cell line treated with different concentrations of NKT2152 under 1.5% O 2 for 24 h (control, 1 μM, and 5 μM), demonstrating mitochondrial ROS levels. (U) Tumor growth curves of xenograft mouse models inoculated with RMG1 cells showing significant antitumor effects of NKT2152 ( n = 15 in control group and n = 13 in treatment group). (V) Tumor growth curves of xenograft mouse models inoculated with KOC7C cells showing significant antitumor effects of NKT2152 ( n = 13, each group). Data are presented as mean ± standard error of the mean. RT‐qPCR data are presented as mean ± standard error of the mean from three independent experiments. * p <.05, ** p <.01, ** p <.001.

Article Snippet: After blocking, the membranes were incubated overnight at 4°C with primary antibodies against HIF2A (D9E3, #7096, 1:1000 dilution, Cell Signaling Technology [CST], RRID: AB_10898028) and ACTB (#4967, CST, RRID: AB_330288 1:1000 dilution, Cell Signaling), followed by HRP‐conjugated secondary antibody (#7074, CST, RRID: AB_2099233,1:3000 dilution).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Protein Concentration, Injection, Fluorescence, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) VHL dependency score in ccRCC cell lines ( N = 21) and other pan cancer lines ( N = 1162). Unpaired Wilcoxon test to calculate significance. The horizontal line in the box marks the median (Q2), the upper and lower hinges correspond to the 25th (Q1) and 75th (Q3) percentiles. The whiskers extend to 1.5× the interquartile range from Q1 to Q3. ( B ) Quantification of normal renal organoid growth (EMKC016) with and without VHL inactivation, N = 32 random growing organoids per condition and time point (mean and S.E.M.). ( C ) Activity of a hypoxia reporter (HRE-ODD-GFP) in normal renal organoids (EMKC016) with and without VHL deletion as determined by fluorescence microscopy. ( D ) Schematic of the pooled CRISPR-Cas9 screening strategy. ( E ) Gene level CRISPR-Cas9-based loss of function screening data. Beta scores showing change in sgRNA construct abundance in VHL mutant cells. N = 2 replicates per condition. ( F ) CRISPR-Cas9 based competition assay in VHL mutant MUT10 cells. HIF1A, HIF2A and ARNT mutant cells competed against cells transduced with non-targeting control constructs (NTC). Two sgRNAs per gene combined, N = 3 technical replicates per condition (mean and S.E.M.). ( G ) VHL mutant human renal epithelial organoids with or without HIF1A inactivation at different time points. ( H ) Quantification of organoid growth from ( G ) over time, N = 13 random growing organoids per condition and time point (mean and S.E.M.). .

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: Activity Assay, Fluorescence, Microscopy, CRISPR, Construct, Mutagenesis, Competitive Binding Assay, Transduction, Control

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) Proliferation of HK2 cells with or without VHL. N = 2 per condition. (mean and S.E.M.). ( B ) Western blot of HIF2A, VHL and Actin on HK2 cells with and without VHL. ( C ) Morphology of HK2 cells with and without VHL. ( D ) Proliferation of VHL mutant HK2 cells with and without VHL re-introduction. N = 3 per condition (mean and S.E.M.). ( E ) Western blot of HIF2A on VHL mutant HK2 cells with and without VHL re-introduction. ( F ) Proliferation of HK2 cells with and without VHL under 5% or 21% O 2 culture conditions. N = 2 per condition (mean and S.E.M.). ( G ) Representative images of human renal epithelial organoids with and without VHL. N = 4 replicates per condition. ( H ) Human renal epithelial organoid proliferation under DMSO or DMOG (3 mM) treatment.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: Western Blot, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) Schematic of doxycycline (dox) inducible VHL re-introduction into VHL mutant (MUT10 and MUT35) and wild-type control (WT8) clones. ( B – D ) Proliferation of WT8 ( B ), MUT10 ( C ) and MUT35 ( D ) cells with and without dox. N = 2 replicates per condition (mean and S.E.M.). ( E ) Western blot of HIF1A, HIF2A, VHL and Actin on WT8, MUT10 and MUT35 cells with and without dox. ( F ) Cas9 editing efficiency tested on WT8, MUT10 and MUT35 cells by a reporter plasmid using fluorescence-activated cell sorting.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: Mutagenesis, Control, Clone Assay, Western Blot, Plasmid Preparation, Fluorescence, FACS

Journal: EMBO Molecular Medicine

Article Title: Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

doi: 10.1038/s44321-025-00361-w

Figure Lengend Snippet: ( A ) CRISPR/Cas9-based genome wide screen data. sgRNA abundance on day 28 relative to start of the assay in VHL mutant clones MUT10 and MUT35. R, Pearson’s correlation coefficient. ( B , C ) As in ( A ) with sgRNAs targeting genes of interest highlighted in red: HIF1A in ( B ) and ARNT in ( C ). ( D ) Pathway enrichment analysis on the top 500 genes the sgRNAs of which are depleted over time in MUT10 and MUT35 cells using the Cancer Hallmarks gene sets. ( E ) A schematic of the competitive proliferation assay. VHL-HIF1A, VHL-HIF2A and VHL-ARNT double mutant cells (BFP labelled) competed against VHL-NTC single mutant cells (GFP labelled). ( F – H ) Western blot of HIF1A, ARNT and HIF2A on MUT10 cells with and without HIF1A, ARNT or HIF2A inactivation, respectively. ( I ) Morphology of VHL-NTC, VHL-HIF1A, VHL-HIF2A and VHL-ARNT cells.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore) and blotted with VHL (BD Pharmingen, 565183, 1:500), HIF1A (Proteintech, 20960-1-AP, 1:1000), HIF2A (Novus Biologicals, NB100-122, 1:1000), beta-actin (Sigma-Aldrich, A1978, 1:30,000), p-ERK (Abcam, ab201015, 1:1000), ERK (Abcam, ab184699, 1:2000) antibodies.

Techniques: CRISPR, Genome Wide, Mutagenesis, Clone Assay, Proliferation Assay, Western Blot

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 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