Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were transfected with either control mimic (con) or miR-30b mimic (30b) and levels of TGFβ1 and TGFβ2 mRNA were assessed by qRT-PCR. Expression levels relative to control mimic transfected cells and normalized to β-actin expression are presented as the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 expression. * P < 0.05, ** P < 0.01 as determined by unpaired Student’s t -test. (B) Cells were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of TGFβ2 protein levels by western blot. β-actin was used as endogenous control. (C) ELISAs for TGFβ1 and TGFβ2 were performed with 24 hour conditioned supernates from HUVECs transfected with 20 nM of either control or miR-30b mimic. Data represents the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 secretion into cell culture supernate. * P = 0.044 as determined by unpaired Student’s t -test. (D) HUVECs were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of Smad2 phosphorylation by western blot.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Cell Culture, Phospho-proteomics

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) JDP2 mRNA expression was assessed in HUVECs transfected with miR-30b mimic (20 nM) as compared to control by qRT-PCR. Data represents the mean ± SEM (n = 3) normalized to β-actin as endogenous control. * P = 0.016 as determined by unpaired Student’s t -test. (B) HUVEC were transfected with 50 nM of either control siRNA or ATF2 siRNA 1 or 2 and RNA was isolated at 48 hours post transfection. Levels of ATF2 and TGFβ2 mRNA were assessed by qRT-PCR with β-actin as endogenous control. Data presented is mean ± SEM (n = 2). Statistically significant decreases in ATF2 and TGFβ2 expression were seen in ATF2 siRNA treated cells as compared to control siRNA treated cells. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by unpaired Student’s t -tests for each ATF2 siRNA compared to control siRNA. (C) Cells transfected with 5 nM of either control siRNA or ATF2 siRNA 1 were seeded onto growth factor reduced BME and the formation of capillary-like cord structures and number of loops was assessed after 24 hours. (D) A statistically significant increase in cord formation was observed in cells depleted of ATF2 through siRNA. Data represents the mean ± SEM (n = 2). * P = 0.041 as determined by unpaired Student’s t -test. (E) HUVECs were co-transfected with miRNA mimic (20 nM) and ATF2 siRNA 1 or 2 (50 nM) in the combinations displayed and cell lysates were collected at 48 hours post transfection and assessed for TGFβ2 mRNA expression. Data presented is mean ± SEM (n = 2). * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA with post hoc analysis. (F) Cells were transfected as in (E) using miRNA mimic (20 nM) and ATF2 siRNA 1 (5 nM) and serum starved overnight in MCDB 131 with 0.5% FBS prior to protein expression analysis by western blot. Data is representative of expression levels observed in two independently performed experiments.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Isolation, Western Blot

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were serum starved overnight in MCDB 131 with 0.5% FBS and stimulated with VEGF (50 ng/ml) in the presence or absence of Avastin (1 μg/ml) for 24 hours. Data represents the mean ± SEM (n = 2) for expression of TGFβ1 and TGFβ2 assessed by qRT-PCR relative to β-actin endogenous control. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA. (B) HUVECs were treated with 5 ng/ml of TGFβ2 for 3 days prior to seeding onto growth factor reduced BME for assessment of capillary-like cord formation after 24 hours. (C) A significant decrease in cord formation is observed in the TGFβ2 treated group. Data represents the mean ± SEM (n = 2). ** P = 0.0072 as determined by unpaired Student’s t -test. (D) HUVECs transfected with 1 nM control or miR-30b mimic were treated 4 hours post transfection with 0.8 μg/ml anti-TGFβ2 neutralizing antibody or rabbit IgG. Media was refreshed after 24 hours, again with rabbit IgG or anti-TGFβ2 antibody and cells were seeded onto growth factor reduced BME 24 hours later (ie. 48 hours post transfection) in media containing rabbit IgG or anti-TGFβ2 antibody. (E) Data represents the mean ± SEM (n = 3) of the number of capillary-like cord structures or number of loops formed after 24 hours on BME. * P < 0.05, ns denotes not significant as determined by ANOVA with post hoc analysis.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A ) Implantation of IL-10-deficient mice with B16/F10 cells resulted in enhanced CD4 + Foxp3 + Tregs in the spleen and tumor on day +15, as assessed by flow cytometry. ( B ) Measurement of TGF-β1 in NP-40-processed B16/F10 tumor tissues (described in the Methods section) obtained from WT and IL-10-deficient tumor-bearing mice. The TGF-β1 cytokine was measured using Luminex (Millipore Merck). ( C ) Among CD4 + T cells from IL10 −/− tumor-bearing mice, splenic TGF-β1 + Foxp3 + cells were increased compared with WTB16/F10 mice; however, differences in the tumor tissues and TDLNs were not significant. ( D ) IL10 deficiency down-regulated Nrp-1 protein expression in tumor tissues. Representative photomicrographs of B16/F10 tumors harvested on day 15. Left panel, representative immunohistochemistry images obtained using a microscope with a 20X objective. Right panel, statistical analysis of the mean IOD for tumor cell proliferation using Image-Pro Plus software. Nonlinear regression was performed using GraphPad Prism (San Diego, CA, USA). ( E ) Spleen Nrp-1-expressing CD4 + Foxp3 + T cells from IL10 −/− tumor-bearing mice were decreased compared with WT B16/F10 mice. Panels ( A,B,D,E ) Evaluated using Student’s t-test to determine statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001between experimental groups. The data are expressed as the mean values ± SEM from n = 3–4 mice. Panel ( C ) Evaluated using ANOVA, ***P < 0.001, ±SEM from n = 3–4 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Flow Cytometry, Luminex, Expressing, Immunohistochemistry, Microscopy, Software

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A ) A high-affinity neutralizing anti-Nrp-1 antibody (described in the Methods section) was used to treat WT or IL10 −/− B16/F10 tumor-bearing mice. The B16/F10 tumor mass volume was monitored every three days after implantation. ( B ) The majority of B16/F10 tumor-derived CD4 + CD8a − lymphocytes were increased in the IL10 −/− mice after treatment with the anti-Nrp-1 polyclonal Ab, as assessed by flow cytometry. ( C ) Use of anti-Nrp-1 in IL10 −/− mice with B16/F10 tumors allowed the augmentation of a CD4 + CD69 + -activated T cell population in the tumor microenvironment by flow cytometry. ( D ) Neutralization of Nrp-1 in IL10 −/− mice implanted with B16/F10 tumors augmented CD4 + IL17A + (Th17) T cells in the tumor microenvironment compared with WT mice with B16/F10 tumors alone, as assessed by flow cytometry. ( E , F ) Numbers within quadrants represent the percentages of positive cells for a given marker within the lymphocyte gate . Anti-Nrp-1 augmented TDLN or tumor-derived CD4 + IFN-γ + T cells in WT or IL10 −/− B16/F10 mice based on flow cytometry. The bar graph shows data from a representative experiment (mean ± SEM; N = 3–4 mice per group) in which two separate experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001; assessed by pairwise multiple comparison procedures (Tukey test) and ANOVA.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Derivative Assay, Flow Cytometry, Neutralization, Marker, Comparison

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A,B ) Measurement of B16/F10 TDLN and tumor-derived CD4 + Foxp3 + Treg cells in IL10 −/− B16/F10 mice or WT B16/F10 mice after treatment with an anti-Nrp-1 polyclonal Ab by flow cytometry. TDLN and tumor tissue samples were processed as described in the Methods section. ( C ) Measurement of B16/F10 tumor-derived Nrp-1 + expression in gated CD4 + Foxp3 + T cells from IL10 −/− B16/F10 mice or WT B16/F10 mice after treatment with the anti-Nrp-1 polyclonal Ab by flow cytometry using micro-dissected and digested tumor tissues. ( D–F ) Measurement of TGF-β, IL6 and VEGF expression in NP-40-processed B16/F10 tumor tissues (described in the Methods section) obtained from mice treated with anti-Nrp-1. The cytokines were measured using Luminex (Millipore Merck). ( G ) Blood vessels stained with an anti-CD31 antibody. The images were obtained using a microscope with a 10X objective. ( H ) Compared with the control group, the tumor tissues of mice treated with the anti-Nrp-1 antibody showed much fewer Ki-67-positive cells. Left panel, representative immunohistochemistry images obtained using a microscope with a 20X objective. Right panel, statistical analysis of the mean IOD for blood vessels and tumor cell proliferation performed using Image-Pro Plus software. Nonlinear regressions were performed using GraphPad Prism (San Diego, CA, USA). The data shown are from one representative experiment of two independent experiments with the same results. The results were evaluated using ANOVA for the determination of statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001 between experimental groups. The data are expressed as the mean values ± SEM from n = 4–6 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Derivative Assay, Flow Cytometry, Expressing, Luminex, Staining, Microscopy, Control, Immunohistochemistry, Software

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A ) A neutralizing anti-TGF-β antibody (described in the Methods section) was used to treat WT or IL10 −/− B16/F10 tumor-bearing mice. The tumor mass volume was monitored every three days after implantation. ( B ) TGF-β1 from NP-40-processed B16/F10 tumor tissues was measured using Luminex. Dual inhibition of IL-10 and TGF-β resulted in significantly lower levels of tumor-secreted VEGF protein in B16/F10 tumor-bearing mice. ( C ) Tumor-derived WT B16/F10-derived Nrp-1 levels were elevated compared with IL10 −/− B16/F10 mice treated with anti-TGF-β or IL10 −/− B16/F10 mice by flow cytometry. ( D ) VEGF from NP-40-processed B16/F10 tumor tissues measured using Luminex. ( E ) The absence of IL-10 or blocked TGF-β antibody had a more pronounced effect on CD4 + CD8a − T cells in B16/F10 tumors. ( F ) Anti-TGF-β in IL10 −/− B16/F10 mice allowed for the expansion of tumor IFN-γ-expressing CD4 + (Th1) populations measured by flow cytometry. ( G ) Anti-TGF-β did not allow the expansion of tumor CD4 + IL17A + (Th17) populations in IL10 −/− B16/F10 mice measured by flow cytometry. The data shown are from one representative experiment of two independent experiments with same results and were evaluated using ANOVA for the determination of statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001between experimental groups. The data are expressed as the mean values ± SEM from n = 4–6 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Luminex, Inhibition, Derivative Assay, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A,B ) Percentages of the different T cell subpopulations gated on lymphocytes (FSC/SSC). IL-10 was more important than TGF-β for increasing the CD4 + Foxp3 + iTreg cell population in the tumor and spleen microenvironment, as measured by flow cytometry. ( C ) The frequency of Nrp-1–expressing Foxp3 + Treg cells from WT or IL10 −/− tumor-bearing mice blocked with or without TGF-β1 was determined within tumors or spleens by flow cytometry gating on CD4 + Foxp3 + cells . These data show that TGF-β increased the ability of tumor-derived Nrp-1 + CD4 + Foxp3 + T cells to support tumor growth. Panel ( A ) Evaluated using Student’s t-test to determine statistical significance with *P < 0.05 between experimental groups. The data are expressed as the mean values ± SEM from n = 3–4 mice. Panels ( B , C ) Evaluated using ANOVA, *P < 0.05, ±SEM from n = 3–4 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Flow Cytometry, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1371/journal.pone.0209786

Figure Lengend Snippet: (A) A flow cytometric analysis showed that PDGFR-α and -β were both detected on C57B/L6 lung fibroblasts. (B) Mouse alveolar type 2 epithelial cells were isolated as described in the Materials and Methods section. The cells obtained were investigated by a FACS analysis. Neither PDGFR-α nor -β was detected in freshly isolated mouse alveolar type 2 epithelial cells. Similar results were obtained at least two independent experiments.

Article Snippet: As the primary antibodies, a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) and a FITC anti-mouse CD326(Ep-CAM) antibody (Biolegend) or anti-alpha smooth muscle actin antibody (abcam, Cambridge, UK) were used as the primary antibodies.

Techniques: Isolation

Journal: PLoS ONE

Article Title: Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1371/journal.pone.0209786

Figure Lengend Snippet: Immunoblotting was performed for the detection of PDGFRs, Akt, and their phosphorylation in lung fibroblasts. Murine lung fibroblasts were stimulated with PDGF-AA (10 ng/ml) and -BB (10 ng/ml) in the presence of the anti-PDGF-α antibody APA5, anti-PDGFR-β antibody APB5, or control Ab. APA5 inhibited the phosphorylation of PDGFR-α induced by PDGF-AA. APB5 inhibited the phosphorylation of PDGFR-β induced by PDGF-BB. The phosphorylation of Akt was also inhibited by both antibodies. Representative immunoblot (A) and corresponding densitometric quantification of PDGFRs phosphorylation and Akt phosphorylation (B-D) are shown.

Article Snippet: As the primary antibodies, a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) and a FITC anti-mouse CD326(Ep-CAM) antibody (Biolegend) or anti-alpha smooth muscle actin antibody (abcam, Cambridge, UK) were used as the primary antibodies.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: PLoS ONE

Article Title: Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1371/journal.pone.0209786

Figure Lengend Snippet: (A)The expression of PDGFRs in the lungs of BLM-treated mice was examined. Staining for PDGFRs was detected in fibrotic lungs. Several cells on alveolar wall showed weak staining for PDGFR-α. PDGFR-β was not clearly detected on alveolar wall. Mesenchymal cells clustering in fibrotic regions showed strong staining for PDGFR-α and -β; however, PDGFR-β was more strongly stained than PDGFR-α. (B) The double-staining with anti-PDGFR antibody and anti-EpCAM or anti-α SMA antibody. PDGFR-α and -β were expressed on α-SMA-positive fibroblasts, not EpCAM-positive lung epithelial cells.

Article Snippet: As the primary antibodies, a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) and a FITC anti-mouse CD326(Ep-CAM) antibody (Biolegend) or anti-alpha smooth muscle actin antibody (abcam, Cambridge, UK) were used as the primary antibodies.

Techniques: Expressing, Staining, Double Staining