human lung fibroblast cells hlf  (ATCC)

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