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: Oncogene

Article Title: TGFbeta1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation.

doi: 10.1038/sj.onc.1203806

Figure Lengend Snippet: Figure 1 In¯uence of TGFb1 on cell proliferation. Treatment of PANC-1 and BxPC-3 cells with TGFb1 resulted in concentration- dependent inhibition of cell proliferation. MIA PaCa-2 cells, characterized by a lack of TGFb receptor type II, were unable to respond to TGFb. In contrast, NIH3T3 ®broblasts showed a marked increase in proliferation after treatment with TGFb1. Cell proliferation was measured by [3H]thymidine incorporation during DNA synthesis. Untreated cells were de®ned as 100%, mean values+s.d. of ®ve independent assays are shown

Article Snippet: Neutralizing TGFb antiserum (AB 101 NA) and anti-TGFb receptor type II (AF 241 NA) were obtained from R&D Systems (Wiesbaden, Germany), antibodies against TGFb receptor type I (T-19), ERK2 (C14), MEK-1 (C-18), Smad2/3 and GST (Z-5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-pan-Ras antibody (Ab-3) was from Oncogene Sciences (Cambridge, MA, USA), anti-b-catenin from Sigma (Deisenhofen, Germany), anti-Smad4 from Transduction Laboratories (San Diego, CA, USA), and polyclonal c-Raf-1 antiserum from Promega (Mannheim, Germany).

Techniques: Concentration Assay, Inhibition, DNA Synthesis

Journal: Oncogene

Article Title: TGFbeta1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation.

doi: 10.1038/sj.onc.1203806

Figure Lengend Snippet: Figure 2 Analyses of TGFb-associated signaling molecules. (a) TGFb receptor type I and II were detected in all cell lines on mRNA level (left panel) and as proteins in membrane fractions (right panel) except for MIA PaCa-2. The apparent molecular weight of TGFbR I is 55 kDa and of TGFbR II 70 kDa. (b) The intracellular TGFb eector protein Smad4 was not detectable in BxPC-3 cells, but clearly visible on mRNA level (left) and as protein in crude extracts (right) of PANC-1 and MIA PaCa-2 carcinoma cell lines

Article Snippet: Neutralizing TGFb antiserum (AB 101 NA) and anti-TGFb receptor type II (AF 241 NA) were obtained from R&D Systems (Wiesbaden, Germany), antibodies against TGFb receptor type I (T-19), ERK2 (C14), MEK-1 (C-18), Smad2/3 and GST (Z-5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-pan-Ras antibody (Ab-3) was from Oncogene Sciences (Cambridge, MA, USA), anti-b-catenin from Sigma (Deisenhofen, Germany), anti-Smad4 from Transduction Laboratories (San Diego, CA, USA), and polyclonal c-Raf-1 antiserum from Promega (Mannheim, Germany).

Techniques: Membrane, Molecular Weight

Journal: Oncogene

Article Title: TGFbeta1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation.

doi: 10.1038/sj.onc.1203806

Figure Lengend Snippet: Figure 4 Stimulation of GDP/GTP exchange on Ras by TGFb1. Treatment of PANC-1, BxPC-3, and NIH3T3 cell with 10 ng/ml EGF induced a markedly elevated amount of Ras-GTP bound to GST-RBD. TGFb1 alone (10 ng/ml) was able to induce Ras activation in all cell lines. Pretreatment with TGFb1 for 15 min followed by addition of 10 ng/ml EGF did not reduce EGF-induced Ras activation. Incubation of cells with TGFb1 and a neutralizing TGFb antibody (nAb) diminished TGFb-induced activation of Ras in PANC-1 cells. (a) (upper panel) Detection of GST-RBD-bound Ras-GTP using pan-Ras antibody. Immunodetection of Ras in cell lysates (30 mg) served as positive control; (lower panel) Detection of GST-RBD with anti-GST antibody to con®rm equal loading. One representative assay out of three is shown. (b) Quanti®cation of Ras activation by densitometric analyses of three independent experiments (mean values+s.e.mean)

Article Snippet: Neutralizing TGFb antiserum (AB 101 NA) and anti-TGFb receptor type II (AF 241 NA) were obtained from R&D Systems (Wiesbaden, Germany), antibodies against TGFb receptor type I (T-19), ERK2 (C14), MEK-1 (C-18), Smad2/3 and GST (Z-5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-pan-Ras antibody (Ab-3) was from Oncogene Sciences (Cambridge, MA, USA), anti-b-catenin from Sigma (Deisenhofen, Germany), anti-Smad4 from Transduction Laboratories (San Diego, CA, USA), and polyclonal c-Raf-1 antiserum from Promega (Mannheim, Germany).

Techniques: Activation Assay, Incubation, Immunodetection, Positive Control

Journal: Oncogene

Article Title: TGFbeta1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation.

doi: 10.1038/sj.onc.1203806

Figure Lengend Snippet: Figure 5 Localization of c-Raf-1 after growth factor and TGFb1 treatment. Distribution of c-Raf-1 in membrane and cytosolic fractions of PANC-1, BxPC-3, and NIH3T3 was detected by Western blot analyses using c-Raf-1 antiserum. Stimulation of quiescent cells with EGF (10 ng/ml) resulted in increased membrane localization in all cell lines. TGFb1 (10 ng/ml) had no eect on membrane association of c-Raf-1 in PANC-1 and BxPC-3 cells, but caused a slight increase in membrane-bound c-Raf-1 in NIH3T3 cells. Cotreatment with EGF and TGFb had the same eect as treatment with EGF alone. Addition of neutralizing TGFb antibody together with TGFb1 served as control. To con®rm equal protein loading, Western blots were reprobed with anti-b-catenin for membrane fractions and anti-ERK2 for cytosolic protein content. One representative assay out of ®ve is shown

Article Snippet: Neutralizing TGFb antiserum (AB 101 NA) and anti-TGFb receptor type II (AF 241 NA) were obtained from R&D Systems (Wiesbaden, Germany), antibodies against TGFb receptor type I (T-19), ERK2 (C14), MEK-1 (C-18), Smad2/3 and GST (Z-5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-pan-Ras antibody (Ab-3) was from Oncogene Sciences (Cambridge, MA, USA), anti-b-catenin from Sigma (Deisenhofen, Germany), anti-Smad4 from Transduction Laboratories (San Diego, CA, USA), and polyclonal c-Raf-1 antiserum from Promega (Mannheim, Germany).

Techniques: Membrane, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Incorporation of a TGF-β2-inhibiting oligodeoxynucleotide molecular adjuvant into a tumor cell lysate vaccine to enhance antiglioma immunity in mice.

doi: 10.3389/fimmu.2023.1013342

Figure Lengend Snippet: FIGURE 1 Effect of TIO3 on the expression of TGF-b2 and MHC-I in cultured GL261 cells or U-251 cells. GL261 or U-251 cells were cultured with TCL, TCL+TIO3 (10 mg/mL) or TCL+cODN (control ODN, 10 mg/mL) for 24 h, respectively, and the mRNA expression of TGF-b2 (A, B) and MHC I (C, D) were measured by qRT-PCR. In addition, GL261 cells were cultured with PMA (100 mg/mL), PMA+TIO3 or PMA+cODN for 48 h, and the protein expression of TGF-b2 (E, F) and MHC I (G, H) were detected by flow cytometry.

Article Snippet: Then, the membranes were blocked with TBST containing 5% nonfat dried milk, shaken at room temperature (22-25°C) for 2 h, and probed with an anti-TGF-b2 antibody (AB-112-NA, R&D Systems, USA), anti-PD-L1 antibody (PA5-20343, Invitrogen) or anti-GAPDH antibody (60004-1-Ig, Proteintech) overnight at 4°C.

Techniques: Expressing, Cell Culture, Control, Quantitative RT-PCR, Cytometry

Journal: Frontiers in immunology

Article Title: Incorporation of a TGF-β2-inhibiting oligodeoxynucleotide molecular adjuvant into a tumor cell lysate vaccine to enhance antiglioma immunity in mice.

doi: 10.3389/fimmu.2023.1013342

Figure Lengend Snippet: FIGURE 5 Effect of TCL+TIO3 on the activation of cytotoxic T lymphocytes and NK cell in glioma-bearing mice. The mice (n=4) were intramuscularly immunized with PBS, TCL, TCL+TIO3 and TCL + cODN in the neck on day 0 and 10, and then and then challenged i.c. with 2×104 GL261 cells on day 14. 4 mice were sacrificed on day 28. (A) Experimental procedure. (B) TGF-b2 expression in glioma tissue isolated from the mice on day 28 by western blotting. (C-E) Percentages of CD4+ T, CD8+ T and NK cells in lymph nodes. (F) Glioma-specific CTL detection. Splenocytes were co-cultured with GL261 cells for 8 h at effector/target ratios of 100:1, 50:1 and 25:1, and methylthiazolyldiphenyl-tetrazolium bromide assays was performed for the cytotoxicity. (G-H) The expression of CD69 and NKG2D on NK cells in lymphocytes. TIO3 plus TCL versus TCL.

Article Snippet: Then, the membranes were blocked with TBST containing 5% nonfat dried milk, shaken at room temperature (22-25°C) for 2 h, and probed with an anti-TGF-b2 antibody (AB-112-NA, R&D Systems, USA), anti-PD-L1 antibody (PA5-20343, Invitrogen) or anti-GAPDH antibody (60004-1-Ig, Proteintech) overnight at 4°C.

Techniques: Activation Assay, Expressing, Isolation, Western Blot, Cell Culture

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Vascular Smooth Muscle Emilin-1 Is a Regulator of Arteriolar Myogenic Response and Blood Pressure

doi: 10.1161/atvbaha.112.254664

Figure Lengend Snippet: Figure 5. Dependence of myogenic response on transforming growth factor (TGF)-β activity in resistance arteries. A, Anti-TGF-β treatment abolished the altered myogenic tone of Emilin1−/− arter ies. Neutralizing antibodies to TGF-β or preimmune IgG (PI IgG) were administered for 24 days to control and Emilin1 mutant mice and the myogenic tone measured. Response to pressure was measured at 25 mm Hg increments, starting from 25 mm Hg and each pressure step was maintained for 20 minutes. Experi mental groups consisted of 5 mice. *P<0.01, Emilin1−/−+PI IgG vs Emilin1+/++PI IgG; §P<0.001, Emilin1−/−+PI IgG vs Emilin1−/−+anti- TGF-β. B, Normalization of myogenic tone of Emilin1−/− vessels by ex vivo treatment with neutralizing antibodies to TGF-β. Resis tance arteries from wild-type and Emilin1−/− mice were mounted in a pressure myograph and neutralizing antibody to TGF-β or PI IgG added to the buffer (0.01 ng/mL). After 30 minutes of incubation, the myogenic response was measured. *P<0.05 vs Emilin1−/−+PI IgG.

Article Snippet: Neutralizing antibodies to TGF-β (0.5 μg/kg per day; R&D Systems Inc., Minneapolis, MN, diluted in PBS) or preimmune IgG were infused for 24 days into Emilin1−/− and eNOS−/− mice or the corresponding wild-type control littermates through osmotic minipumps (Alzet model 2004, Durect Corporation, Cupertino, CA) implanted subcutaneously on the right side of the back of the mice.

Techniques: Activity Assay, Control, Mutagenesis, Ex Vivo, Incubation