Journal: bioRxiv

Article Title: Wnt1-Cre mediated deletion of BMP7 suggests a role for neural crest-derived BMP7 in retina development and function

doi: 10.1101/2021.11.03.466838

Figure Lengend Snippet: Bmp7 ctrl (A, C, E, G, G’, I, K) and Bmp7 ncko (B, D, F, H, H’, J, L) retina were stained for various neuronal proteins and DAPI for nuclei (A-B) using immunofluorescence. Reduced expression of CALB (C-D) was observed in GCL and INL of the mutant retina. Increased expression of NF-H (E-F) was observed in the GCL of Bmp7 ncko . A slight increase of GFAP (G-H) expression was observed in the GCL. (G’-H’) Higher magnification images of the GFAP expression in the GCL. Expression of IBA1 (I-J) was increased in GCL, INL and ONL layers of Bmp7 ncko . Expression of PAX6 (K-L) was also reduced in the GCL and INL of the mutant mice. Immunostaining was performed on paraffin-embedded P30 retina (n=3/genotype). CALB: calbindin; NF-H: neurofilament heavy; GFAP: glial fibrillary acidic protein; IBA1: Ionized calcium binding adaptor molecule 1; PAX6: paired box protein 6. gcl: ganglion cell layer; ipl: inner plexiform layer; inl: inner nuclear layer; opl: outer plexiform layer; onl: outer nuclear layer; is/os: inner segments/outer segments; rpe: retinal pigment epithelium. P30: postnatal day 30.

Article Snippet: Primary antibodies used were: Calbindin (Calb; Santa Cruz, sc28285), Neurofilament Heavy (NF-H; Abcam, ab187374), Glial Fibrillary Acidic Protein (Gfap; Abcam, ab4674), Pax6 (prb-278P), Rhodopsin (Rho; Abcam, ab98887), Recoverin (Rcvrn; Abcam, ab5585), Blue Opsin (S-op; short-wavelength; Millipore Sigma, ab5407), Red & Green Opsin (R&G-op; medium wavelength; Millipore Sigma, ab5405), Tomato Lectin (Invitrogen L32470 DyLight 488), Bone Morphogenetic Protein 7 (Bmp7; Abcam, ab84684) and Green Fluorescent Protein (Gfp; Abcam, ab6556, provided by Luc Berthiaume, Department of Cell Biology, University of Alberta).

Techniques: Staining, Immunofluorescence, Expressing, Mutagenesis, Immunostaining, Binding Assay

Journal: bioRxiv

Article Title: Wnt1-Cre mediated deletion of BMP7 suggests a role for neural crest-derived BMP7 in retina development and function

doi: 10.1101/2021.11.03.466838

Figure Lengend Snippet: (Top panel) A summary of aberrant proteins expression observed in P30 Bmp7 ncko mice retina. Increased expression of proteins in green and proteins with decreased expression in red. BMP7 expression in blue. (Bottom panel) Data mining of single cell RNA-Seq confirmed dynamic expression pattern of BMP7 during development along with co-expression of proteins (CALB, NF-H, PAX6, RHO, S-OP, RCVRN) assessed in this study. E: embryonic day; P: postnatal day. RHO: rhodopsin; RCVRN: recoverin; S-OP: blue opsin; CALB: calbindin; NF-H: neurofilament heavy; PAX6: paired box protein 6.

Article Snippet: Primary antibodies used were: Calbindin (Calb; Santa Cruz, sc28285), Neurofilament Heavy (NF-H; Abcam, ab187374), Glial Fibrillary Acidic Protein (Gfap; Abcam, ab4674), Pax6 (prb-278P), Rhodopsin (Rho; Abcam, ab98887), Recoverin (Rcvrn; Abcam, ab5585), Blue Opsin (S-op; short-wavelength; Millipore Sigma, ab5407), Red & Green Opsin (R&G-op; medium wavelength; Millipore Sigma, ab5405), Tomato Lectin (Invitrogen L32470 DyLight 488), Bone Morphogenetic Protein 7 (Bmp7; Abcam, ab84684) and Green Fluorescent Protein (Gfp; Abcam, ab6556, provided by Luc Berthiaume, Department of Cell Biology, University of Alberta).

Techniques: Expressing, RNA Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Fecal-adherent mucus is a non-invasive source of primary human MUC2 for structural and functional characterization in health and disease

doi: 10.1016/j.jbc.2024.105675

Figure Lengend Snippet: Analysis of MUC2 glycosylation on fecal sections in situ . A , schematic of glycan structures recognized by the lectins used in this figure to assist in interpretation. B , Tiled cross-section of healthy human feces dual stained with Wheat Germ Agglutinin (WGA) and Aleuria aurantia lectin (AAL). C and D , high-mag imaging human mucus stained for Maa c kia ammurensis (MAL)-II and Sambucus nigra (SNA)-1 ( B ), and Lycopersicon esculentum (Tomato) lectin (LEL) and Ulex europaeus -1 (UEA-1) ( C ). E , Lectin blotting of fecal MUC2. Lanes that are close together represent samples run on the same gel and transferred to the same membrane, but separated for specific blots. White space indicates lanes were not adjacent. Bar on the right of blot = 250 kDa maker. F , dual stain for MUC2 and MALII to analyze MALII distribution in human versus mouse fecal sections. Results are representative of five individual fecal sections.

Article Snippet: The lectins used were biotinylated A. aurantia lectin (AAL, five ug/ml in ADB, B-1395–1, Vector Laboratories), Wheat-Germ Agglutinin-Fluorescein (WGA-Fluorescein 5 ug/ml in ADB, FL-1021, Vector Laboratories), biotin or FITC-conjugated Tomato lectin (5 ug/ml in ADB, B-1175–1, Vector Laboratories); Biotinylated or Rhodamine/DyLight 350/DL-647-conjugated U. europaeus agglutinin I (UEA1, 4 ug/ml, RL-1062–2 and others, Vector Laboratories), and biotinylated or FITC- S. nigra agglutinin 1 (SNA-1, 10 ug/ml, FL-1301–2, Vector laboratories), biotinylated M. amurensis lectin II (MALII, 3 ug/ml, B-1265–1, Vector Laboratories), and/or polyclonal anti-MUC2 ( ).

Techniques: In Situ, Staining, Imaging, Membrane

Journal: Cellular and Molecular Bioengineering

Article Title: Injectable, Hyaluronic Acid-Based Scaffolds with Macroporous Architecture for Gene Delivery

doi: 10.1007/s12195-019-00593-0

Figure Lengend Snippet: Macrophages, fibroblasts and endothelial cells are found to infiltrate scaffolds. F4/80+ cells were present throughout NP-HA, PEG-MP, and HA-MP scaffolds (a). F4/80+ intensity/area was statistically equivalent for all scaffolds (b). ERTR7+ cells were also present within all scaffolds (c) with significantly less intensity/area in NP-HA than in HA-MP scaffolds (d). PEG-MP scaffolds had visually greater immunostaining and intensity/area for ERTR7 than NP-HA scaffolds but were not significantly different than NP-HA or HA-MP scaffolds. CD31+ cells were most notably present in HA-MP scaffolds, with some presence in PEG-MP scaffolds (e). Significantly greater CD31+ intensity/area was observed in HA-MP compared to NP-HA, but not PEG-MP, scaffolds (*p < 0.05, Kruskal–Wallis test with Dunn’s post-hoc tests, n = 4–5) (f). CD31+ staining is clear at depths of over 200 µm in HA-MP but not PEG-MP or NP-HA scaffolds (G). Scaffolds were initially loaded with 1.3 × 109 active lentiviral particles per NP-HA or HA-MP scaffold, and 9.8 × 108 per PEG-MP scaffold. Scaffolds are indicated on the left-hand side of the dashed lines. Scale bars = 200 µm, 20 µm for zoomed inset. Arrows indicate F4/80, ERTR7, or CD31 positive cells within scaffolds.

Article Snippet: Primary antibodies used were rat anti-F4/80 (1:200, MCA497R, AbD Serotec) to detect macrophages, rat anti-ERTR7 (1:200, sc-73355, Santa Cruz Biotechnology) to detect fibroblasts, rat anti-CD31 (1:200, BDB553370, BD Biosciences) to detect endothelial cells, and goat anti-td tomato (1:200, LS-C340696, LifeSpan BioSciences, Inc.) to detect transduced cells.

Techniques: Immunostaining, Staining

Journal: Cellular and Molecular Bioengineering

Article Title: Injectable, Hyaluronic Acid-Based Scaffolds with Macroporous Architecture for Gene Delivery

doi: 10.1007/s12195-019-00593-0

Figure Lengend Snippet: Cells expressing the td-tomato transgene are located within scaffolds. Td-tomato expression overlapped with F4/80+, ERTR7+, and CD31+ positive areas, in order of with decreasing abundance. Arrows indicate F4/80, ERTR7, or CD31 positive cells which express td-tomato. Scaffolds were initially loaded with 3.5 × 108 active lentiviral particles. Scaffolds are located on the left-hand side of the dashed lines. Zoomed insets show examples of double-positive cells (RGB overlays and separate red and green channels). Scale bars = 200 µm, 10 µm for zoomed insets (n = 2).

Article Snippet: Primary antibodies used were rat anti-F4/80 (1:200, MCA497R, AbD Serotec) to detect macrophages, rat anti-ERTR7 (1:200, sc-73355, Santa Cruz Biotechnology) to detect fibroblasts, rat anti-CD31 (1:200, BDB553370, BD Biosciences) to detect endothelial cells, and goat anti-td tomato (1:200, LS-C340696, LifeSpan BioSciences, Inc.) to detect transduced cells.

Techniques: 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: ANGPTL4 is upregulated in senescent cells. (A) Venn diagram showing genes commonly upregulated between 6 different senescence transcriptome datasets: RAS‐induced senescence in IMR90 fibroblasts (IMR_RAS); cigarette smoke condensate‐induced senescence in MRC5 fibroblasts (MRC5_CSC); etoposide‐induced senescence in WI38 fibroblasts (WI38_ET); human umbilical vein endothelial cells in replicative senescence (HUVEC_RS); MEK‐induced senescence in human mammary epithelial cells (hMEC_MEK); and H 2 O 2 ‐induced senescence in astrocytes (Astrocytes_H 2 O 2 ). ANGPTL4, the only gene upregulated in all conditions is indicated. (B) Relative mRNA expression of P21 , KI67 and ANGPTL4 genes by using RT‐qPCR, in MRC5/RAF:ER cells untreated (CTRL) or treated with 4‐OHT (RAF) for Oncogene Induced Senescence (OIS), in p20‐p23 (EARLY) and p38‐p41 (LATE) MRC5 passages for replicative senescence (RS), and in MRC5 untreated (NT) or treated with bleomycin (BLEO) for therapy‐induced senescence (TIS). Mean ± SEM of n = 3 independent experiments. Paired t‐test results are indicated. (C) Western blot analysis of ANGPTL4 expression in MRC5/RAF:ER not treated (CTRL) or treated with 4‐OHT (OIS). Full‐length ANGPTL4 detected and TUBULIN loading control in whole extract and cleaved ANGPTL4 (c‐ANGPTL4) detected in cell supernatants. Representative image 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: Expressing, Quantitative RT-PCR, Western Blot, Control

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 production of the proinflammatory SASP during senescence. (A) RT‐qPCR analysis of ANGPTL4 and other SASP members without RAF activation (NT) or at different time points after RAF activation, from day 1 (D1) to day 5 (D5), in MRC5/RAF:ER cells. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA tests are shown. (B–D) MRC5/RAF:ER cells were transfected with control (siCTRL) or ANGPTL4 (siANGPTL4) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS). (B) RT‐qPCR analysis of the gene expression of SASP members. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA tests are shown. (C) Western blot analysis of ANGPTL4 and IL6 proteins. Representative image of n = 3 independent experiments. (D, E) Quantification of ANGPTL4 (D) and of IL1A, IL6 and IL8 SASP factors (E) performed by ELISA. Mean protein concentrations measured in cell culture medium are ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are indicated.

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: Quantitative RT-PCR, Activation Assay, Transfection, Control, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

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 acts upstream of IL1A to activate the transcriptional proinflammatory SASP program. (A) MRC5 cells were transfected with control (siCTRL) or siRNA targeting IL1A (siIL1A) during seeding. The days after, cells were infected with control or ANGPTL4 encoding retroviral vectors. Three days later, RNAs were prepared. Relative mRNA expression of ANGPTL4 and proinflammatory SASP member genes was measured by RT‐qPCR. (B) MRC5 cells were transfected with control (siCTRL) or siRNA targeting RELA (siRELA) or ANGPTL4 (siANGPTL4) during seeding. The days after, cells were infected with control or IL1A encoding lentiviral vectors. Three days later, RNAs were prepared. Relative mRNA expression of ANGPTL4, RELA and proinflammatory SASP member genes was measured by RT‐qPCR Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown.

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: Transfection, Control, Infection, Retroviral, Expressing, Quantitative RT-PCR

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: Knockdown of ANGPTL4 inhibits the ability of the SASP to induce activation of human neutrophils, a mark of inflammation. (A, B) Supernatants of MRC5/RAF:ER cells transfected with control (siCTRL), ANGPTL4 (siANGPTL4) or RELA (siRELA) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS) were applied to primary human neutrophils. (A) Analysis of CD63 neutrophils degranulation marker. A representative flow cytometry profile is shown. Histograms on the right panel are the quantification of MFI. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown. (B) Analysis of STAT5 pathway activation according to phosphorylation of STAT5. The left panel displays a representative flow cytometry profile. Histograms on the right panel are the quantification of MFI (Mean Fluorescence Intensity). Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown.

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: Knockdown, Activation Assay, Transfection, Control, Marker, Flow Cytometry, Phospho-proteomics, Fluorescence

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: Cleavage of ANGPTL4 by FURIN is required for its ability to promote the proinflammatory SASP. (A‐C) MRC5/RAF:ER cells were transfected with control (siCTRL) or FURIN (siFURIN) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS). (A) RT‐qPCR analysis against the indicated genes 3 days after 4‐OHT treatment. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown. (B) Western blot analysis of FURIN, ANGPTL4: Full length in whole cell extracts (ANGPTL4) and secreted in the supernatant (cANGPTL4) and IL6. Representative picture of n = 3 independent experiments. (C) RT‐qPCR analysis of the indicated SASP encoding genes. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test values are shown. (D) Relative mRNA expression of FURIN , ANGPTL4 and proinflammatory SASP encoding genes measured by RT‐qPCR in MRC5 cells overexpressing or not ANGPTL4 (ANGPTL4 OE) and transfected with control (siCTRL) or FURIN (siFURIN) siRNA. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown.

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: Transfection, Control, Quantitative RT-PCR, Western Blot, 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: 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: Frontiers in Molecular Neuroscience

Article Title: Analysis of the pathogenicity and pathological characteristics of NOTCH3 gene-sparing cysteine mutations in vitro and in vivo models

doi: 10.3389/fnmol.2024.1391040

Figure Lengend Snippet: Pathological changes in R75Q mouse under electron microscope. L = lumen of blood vessels, EC = endothelial cell, BM = basement membrane, VSMC = vascular smooth muscle cell, M = mitochondrion, black arrows and white * mark GOM.

Article Snippet: Lycopersicon Esculentum (Tomato) Lectin (Thermo, United States) was used to label vascular endothelial cells and transgelin/SM22 Polyclonal antibody (Proteintech, United States) was used to label smooth muscle cells.

Techniques: Microscopy, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Analysis of the pathogenicity and pathological characteristics of NOTCH3 gene-sparing cysteine mutations in vitro and in vivo models

doi: 10.3389/fnmol.2024.1391040

Figure Lengend Snippet: Results of lectin and TUNEL immunofluorescence staining of R75Q mice brain. (A) Immunofluorescence staining exhibits endothelial cells (red), SMC (green) and nuclei (blue) in mice brain. (B) Semi-quantitative statistical analysis, endothelial cell coverage in different experimental groups. (C) The positive cells in both groups were counted and statistically analyzed, and the data were finally normalized to the control group. (D) Immunofluorescence staining exhibits dead cells (red), and nuclei (blue) in mice brain. Data are presented as means ± SD. ∗ p < 0.05, ns p > 0.05, n = 4 per group. One—way ANOVA or Kruskal–Wallis H test, and Least-Significant Difference ( LSD ) was used for pairwise comparisons.

Article Snippet: Lycopersicon Esculentum (Tomato) Lectin (Thermo, United States) was used to label vascular endothelial cells and transgelin/SM22 Polyclonal antibody (Proteintech, United States) was used to label smooth muscle cells.

Techniques: TUNEL Assay, Immunofluorescence, Staining, Control

Journal: Oncotarget

Article Title: Compound genetically engineered mouse models of cancer reveal dual targeting of ALK1 and endoglin as a synergistic opportunity to impinge on angiogenic TGF-β signaling

doi: 10.18632/oncotarget.12604

Figure Lengend Snippet: ( A ) Representative images of Podocalyxin-stained vessels in PanNETs of 12-week old RIP-TAg2 mice. Scale bar 50 μm. B-E Vessel analysis of Podocalyxin-stained vessels in PanNETs of 12-week old RIP-TAg2 mice; total vessel area ( B ), average vessel length ( C ), total vessel length ( D ), and total number of junctions ( E ). n = 2–3 mice Data are mean ± SEM. * P < 0.05; ** P < 0.01 vs. Wildtype with Student's t-test

Article Snippet: Primary antibodies used in this study were Podocalyxin (dilution 1:100; R&D Systems, AF1556), αSMA-Cy3 (Clone 1A4; dilution 1:100; Cy3 conjugated; Sigma, C6198), and Isolectin IB4 (dilution 1:50; AF488 conjugated; Thermo Fisher, I21411).

Techniques: Staining

Journal: Oncotarget

Article Title: Compound genetically engineered mouse models of cancer reveal dual targeting of ALK1 and endoglin as a synergistic opportunity to impinge on angiogenic TGF-β signaling

doi: 10.18632/oncotarget.12604

Figure Lengend Snippet: ( A ) Representative images of Podocalyxin-stained vessels in PanNETs of 12-week old RIP-TAg2 mice. Vessel junctions are highlighted in second row with white dots. Scale bar 20 μm. ( B ) Representative images of Lectin-FITC perfused vessels in PanNETs of 12-week old RIP-TAg2 mice. Scale bar 50 μm. ( C ) High-magnification vessel details in PanNETs stained with Isolectin GS-IB4. Scale bar 20 μm. ( D – H ) Analysis of vessel features in Podocalyxin-stained PanNETs from RIP1-TAg2 mice; total vessel area (C), average vessel length (D), total number of junctions (E), junction density (F), and lacunarity (G). ( I ) Quantification of immunofluorescence of FITC-conjugated Lectin perfusion relative to Podocalyxin expression. Data are mean ± SEM. * P < 0.05 vs. Wildtype with Student's t-test .

Article Snippet: Primary antibodies used in this study were Podocalyxin (dilution 1:100; R&D Systems, AF1556), αSMA-Cy3 (Clone 1A4; dilution 1:100; Cy3 conjugated; Sigma, C6198), and Isolectin IB4 (dilution 1:50; AF488 conjugated; Thermo Fisher, I21411).

Techniques: Staining, Immunofluorescence, Expressing

Journal: Oncotarget

Article Title: Compound genetically engineered mouse models of cancer reveal dual targeting of ALK1 and endoglin as a synergistic opportunity to impinge on angiogenic TGF-β signaling

doi: 10.18632/oncotarget.12604

Figure Lengend Snippet: ( A – B ) qRT-PCR expression levels of downstream products of ALK1 and ALK5 signaling (A), and Notch signaling (B) from LYVE1-/CD31+ endothelial cells isolated from PanNET tumors. Expression levels are relative to L19 housekeeping gene. ( C ) Representative images of labelled PanNET tumors from RIP1-TAg2 mice, showing Podocalyxin (green), α-Smooth Muscle Actin (red) and nuclei (DAPI, blue). Scale bar 50 μm. ( D ) Quantification of positively-stained pixels of α-Smooth Muscle Actin relative to Podocalyxin in PanNET tumors. n = 4 mice per group. ( E ) Number of individual hepatic micrometastases in RIP1-TAg2 mice at 12 weeks. SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. Wildtype with Student's t-test .

Article Snippet: Primary antibodies used in this study were Podocalyxin (dilution 1:100; R&D Systems, AF1556), αSMA-Cy3 (Clone 1A4; dilution 1:100; Cy3 conjugated; Sigma, C6198), and Isolectin IB4 (dilution 1:50; AF488 conjugated; Thermo Fisher, I21411).

Techniques: Quantitative RT-PCR, Expressing, Isolation, Staining

Journal: Journal of the American Society of Nephrology

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/asn.2015080877

Figure Lengend Snippet: Figure 2. RAAS inhibition increases the number of labeled cells of renin lineage in the juxtaglomerular compartment in non-diseased mice renin/red fluorescent protein co- staining. Confocal microscopy showing co-staining for renin (green color, first column) and the RFP reporter (red color, middle column) as single panels. A merge of the two panels creates a yellow color if renin and RFP staining co-localize (right column). Nuclei stain

Article Snippet: To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Labeling, Staining, Confocal Microscopy

Journal: Journal of the American Society of Nephrology

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/asn.2015080877

Figure Lengend Snippet: Figure 3. RAAS inhibition increases the number of labeled cells of renin lineage in the juxtaglomerular compartment and migration of fate-mapped cells of renin lineage in FSGS mice (A–C) Shows confocal images of co-staining for renin (green, first column) and the RFP reporter (red, middle column), and when the images were merged (right column); (D, E) shows quantitative data. Following the induction of experimental FSGS in Ren1cCreERxRs-tdTomato-R mice, diseased mice were randomized on day 3 to

Article Snippet: To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Labeling, Migration, Staining

Journal: Journal of the American Society of Nephrology

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/asn.2015080877

Figure Lengend Snippet: Figure 4. RAAS inhibition increases the number of labeled CoRL in the JGC and migration of fate-mapped CoRL in FSGS mice. (A–C) Confocal microscopy of co-staining of BrdU (green, left column), red fluorescent protein (RFP, red, middle column) and DAPI (blue), and merged images (yellow, right column) (A) Enalapril in non-diseased mice: (A, i) BrdU stained cell detected in the JGC; (A, ii) RFP stained cells in the JGC; (A, iii) BrdU stained cell co-localizes with RFP+ cell in the JGC (arrow head).

Article Snippet: To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Labeling, Migration, Confocal Microscopy, Staining

Journal: Journal of the American Society of Nephrology

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/asn.2015080877

Figure Lengend Snippet: Figure 5. CoRL express podocyte proteins and show ultrastructural features of po- docytes in CoRL reporter mice with experimental FSGS after RAAS inhibition. (A) Confocal microscopy of double-staining for synaptopodin (synpo, green) and RFP (red). (A, i) in FSGS mouse at day 28, Synpo staining is restricted to podocytes; (A, ii) RFP staining detected within glomerulus (inset); (A, iii) Synpo and RFP merge (yellow, ar- rowhead) in the glomerulus. (A, iv–vi) Higher-power magnification of insets shown above. Note the morphology of the RFP+CoRL in the glomerulus in (A, v) that re- sembles characteristic podocyte features. (B) Confocal microscopy of WT-1 (green) and RFP (red) staining. (B, i) WT-1 staining is detected in the glomerulus in the nucleus in a typical podocyte distribution, and an occasional WT-1–stained cell is detected in the cytoplasm in the JGC. (B, ii) RFP staining is detected in the JGC and IGC. (B, iii) The merge shows that cells in the inset costain for nuclear WT-1 and for RFP. (B, iv–vi) High power of the insets above. Arrowhead shows example of cell in the glomerulus costaining for nuclear WT-1 and RFP. (C–E) Transmission electron micrograph of RFP- stained cells: (C) Renin granules are noted (black arrows). Arrowheads show RFP- labeled CoRL. (D) RFP does not colocalize with podocytes. (E) Subset of RFP stained cells have foot processes (arrowheads) characteristic of podocytes.

Article Snippet: To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Confocal Microscopy, Double Staining, Staining, Transmission Assay, Labeling

Journal: Journal of the American Society of Nephrology

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/asn.2015080877

Figure Lengend Snippet: Figure 6. CoRL express PEC and mesangial cell proteins in CoRL reporter mice with experimental FSGS after RAAS inhibition. (A) Confocal microscopy of PAX8 (green) and RFP (red) costaining. (A, i) PAX8 staining detected in classic PEC distribution along Bowman’s capsule. (A, ii) RFP-stained cell along Bowman’s capsule within inset box. (A, iii) Cell along Bowman’s capsule costaining for RFP and PAX8. (A, iv–vi) Higher- power magnification of insets above. (B) Confocal microscopy of (B, i) a8 integrin staining detected in a classic mesangial cell distribution. (B, ii) RFP-stained cells in the JGC and in the glomerulus. (B, iii) Cell costaining for a8 integrin and RFP. (B, iv–vi) Higher-power magnification of insets above.

Article Snippet: To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Confocal Microscopy, Staining