Journal: Cells

Article Title: Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells

doi: 10.3390/cells8010023

Figure Lengend Snippet: Collagen synthesis. ( A ) Basal collagen synthesis. ( B ) Collagen synthesis after TGF-β (10 nM) stimulation for 24 h. Measurement based on incorporation of [ 3 H]-proline into collagen. Data are mean ± SEM of triplicate determinations. ( C ) Cells treated with or without TGF-β for 24 h were lysed and proteins subjected to immunoblotting using antibodies against TGF-β receptor I and II, phospho-Smad-2, Smad-2, phospho-Smad-3, and Smad-3. GAPDH was used as loading control. * p < 0.05, ** p < 0.01 comparing average of hPSCs with HPaSteC, i-mPSCs for ( A ); ** p < 0.01 comparing control (non-treated) cells with TGF-β treated cells for ( B ). PSC, pancreatic stellate cell; hPSC, human primary PDAC-derived PSC culture; HPaSteC, PSCs from normal human pancreas; i-hPSC, immortalized human PSCs; i-mPSC C2 and C3, immortalized mouse PSCs clone 2 and 3.

Article Snippet: Reagents were purchased from the following sources: BODIPY™ 493/503, Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, DMEM F-12 containing GlutaMAX-I, penicillin-streptomycin (Pen-Strep), amphotericin B, trypsin/EDTA, fetal bovine serum (FBS), and Pierce TM BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA); bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), gemcitabine hydrochloride, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), phosphate buffered saline (PBS), and senescence cells histochemical staining kit from Sigma–Aldrich (St. Louis, MO, USA); TGF-β from R&D Systems Europe (Abingdon, UK); human anti-alpha smooth muscle actin (α-SMA; BS66) from Nordic Biosite AB (Taby, Sweden); anti-glial fibrillary acidic protein (GFAP) (GA5), anti-epithelial cell adhesion molecule (EpCAM; VU1D9), anti-vimentin (D21H3), anti-GAPDH and Smad2/3 antibody sampler kit from Cell Signaling Technology (Beverly, MA, USA); anti- TGF-β receptor I (TGF-β RI) and anti-TGF-β receptor II (TGF-β RII) from Abcam (Cambridge, UK); secondary HRP-conjugated antibodies goat anti-mouse and goat anti-rabbit IgG from Bio-Rad Laboratories (Hercules, CA, USA); secondary Alexa Fluor-conjugated antibodies (anti-mouse and anti-rabbit) and DAPI from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Western Blot, Derivative Assay

Journal: Cells

Article Title: Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells

doi: 10.3390/cells8010023

Figure Lengend Snippet: Characteristics of the various pancreatic stellate cell (PSC) cultures.

Article Snippet: Reagents were purchased from the following sources: BODIPY™ 493/503, Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, DMEM F-12 containing GlutaMAX-I, penicillin-streptomycin (Pen-Strep), amphotericin B, trypsin/EDTA, fetal bovine serum (FBS), and Pierce TM BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA); bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), gemcitabine hydrochloride, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), phosphate buffered saline (PBS), and senescence cells histochemical staining kit from Sigma–Aldrich (St. Louis, MO, USA); TGF-β from R&D Systems Europe (Abingdon, UK); human anti-alpha smooth muscle actin (α-SMA; BS66) from Nordic Biosite AB (Taby, Sweden); anti-glial fibrillary acidic protein (GFAP) (GA5), anti-epithelial cell adhesion molecule (EpCAM; VU1D9), anti-vimentin (D21H3), anti-GAPDH and Smad2/3 antibody sampler kit from Cell Signaling Technology (Beverly, MA, USA); anti- TGF-β receptor I (TGF-β RI) and anti-TGF-β receptor II (TGF-β RII) from Abcam (Cambridge, UK); secondary HRP-conjugated antibodies goat anti-mouse and goat anti-rabbit IgG from Bio-Rad Laboratories (Hercules, CA, USA); secondary Alexa Fluor-conjugated antibodies (anti-mouse and anti-rabbit) and DAPI from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Isolation, Gradient Centrifugation, Transfection, Expressing, DNA Synthesis, Migration

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: PeerJ

Article Title: CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia

doi: 10.7717/peerj.9796

Figure Lengend Snippet: To determine the role of miR-152-3p in CFs, CFs were transfected with the miR-152-3p mimic or inhibitor. (A–G and J–P) Representative images of EdU-stained CFs from different groups. Quantification of EdU+ cells presented as the % EdU-positive cells and Hoechst-stained nuclei. The orange color is EdU-positive cells, and the blue color is nuclei stained by Hoechst 33342. Scale bar = 20 μm. n = 10. * P < 0.05, vs. MNC group or INC group. (H, I and Q, R) The protein expression levels of Col I and Col III were measured by western blot analysis. n = 3. * P < 0.05, vs. MNC group or INC group. (S) Relative expression of miR-152-3p in CFs after transfection with miR-152-3p mimic or inhibitor was examined by RT-qPCR analysis. n = 3. * P < 0.05, vs. hypoxia group. (T) TGF-β2 was predicted as a target gene of miR-152-3p using the TargetScan database. HEK-293T cells were cotransfected with RLuc-TGF-β2-WT or RLuc-TGF-β2-Mut and miR-152-3p mimics or NC mimic. Luciferase activity was detected using the dual luciferase reporter assay at 48 h post-transfection. n = 3. * P < 0.05, vs. TGF-β2-WT + NC group. # P < 0.05, vs. TGF-β2-WT + miR-152-3p group. (U–X) The protein expression level of TGF-β2 was measured by western blot analysis. n = 3. * P < 0.05, vs. MNC group or INC group.

Article Snippet: The primary anti-bodies: Anti-Collagen I (Cell Signaling Technology, 91144, dilution: 1:1,000), Anti-Collagen III (Abcam, ab7778, dilution: 1:7,000), Anti-α-SMA (Cell Signaling Technology, 19245, dilution: 1:1,000), Anti-p-Smad2 (Cell Signaling Technology, 18338, dilution: 1:1,000), Anti-p-Smad3 (Cell Signaling Technology, 9520, dilution: 1:1,000), Anti-TGF-β2 (Bioss, bs-20412R, dilution: 1:1,000) and Anti-β-actin Antibody (Abcam, ab8227, dilution: 1:1,000), were used.

Techniques: Transfection, Staining, Expressing, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Reporter Assay

Journal: PeerJ

Article Title: CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia

doi: 10.7717/peerj.9796

Figure Lengend Snippet: (A–E) The cell cycle was investigated by a cell cycle assay in cardiac fibroblasts. The results represent the percentage of cells in G1 and S + G2/M phases for the indicated conditions. n = 3. * P < 0.05, # P < 0.05. (F–J) Wound scratch assay for the different groups. The average sizes of the gaps were measured at 48 h. Scale bar = 200 μm. n = 5. * P < 0.05, # P < 0.05. (K and L) The protein expression levels of Col I, Col III, TGF-β2, p-Smad2 and p-Smad3 were measured by western blot analysis. n = 3. * P < 0.05, # P < 0.05. * P < 0.05, vs. hypoxia group, # P < 0.05, vs. si-circHIPK3 group.

Article Snippet: The primary anti-bodies: Anti-Collagen I (Cell Signaling Technology, 91144, dilution: 1:1,000), Anti-Collagen III (Abcam, ab7778, dilution: 1:7,000), Anti-α-SMA (Cell Signaling Technology, 19245, dilution: 1:1,000), Anti-p-Smad2 (Cell Signaling Technology, 18338, dilution: 1:1,000), Anti-p-Smad3 (Cell Signaling Technology, 9520, dilution: 1:1,000), Anti-TGF-β2 (Bioss, bs-20412R, dilution: 1:1,000) and Anti-β-actin Antibody (Abcam, ab8227, dilution: 1:1,000), were used.

Techniques: Cell Cycle Assay, Wound Healing Assay, Expressing, Western Blot

Journal: Nutrients

Article Title: Dietary Soy Protein Isolate Attenuates Intestinal Immunoglobulin and Mucin Expression in Young Mice Compared with Casein

doi: 10.3390/nu12092739

Figure Lengend Snippet: Primers for qRT-PCR.

Article Snippet: The following antibodies were used: anti-Janus kinase-1 (JAK1) (bs-1439R, Bioss, Beijing, China), anti-phospho (p)-JAK1 (Tyr1034 + Tyr1035, bs-3238R, Bioss, China), anti- signal transducer and activator of transcription 6 (STAT6) (380957, ZEN BIO, China), anti-p-STAT6 (Tyr641, ab263947, Abcam, Cambridge, MA, USA), anti-pIgR (AF2800-SP, R&D Systems, Minneapolis, MN, USA), anti-TGFβ1 (bs-4538R, Bioss, China), and anti-β-actin (AP0060, Bioworld Technology Inc., Bloomington, MN, USA).

Techniques:

Journal: Nutrients

Article Title: Dietary Soy Protein Isolate Attenuates Intestinal Immunoglobulin and Mucin Expression in Young Mice Compared with Casein

doi: 10.3390/nu12092739

Figure Lengend Snippet: Effect of SPI on JAK1-STAT6 signaling and TGFβ levels in mouse ileum tissue. ( A ) qRT-PCR analysis of Jak1 , Stat6 , Tgfb1 , and Tgfb2 mRNA expression levels in mouse ileum tissue (normalized against Actb (β-actin); n = 8). ( B ) Western blotting analysis of JAK1, p -JAK1, STAT6, p -STAT6, pIgR, and TGFβ1 protein levels in mouse ileum tissue ( n = 6). ( C ) The statistical analyses of the Western blotting results. Results are normalized to β-actin ( n = 6). All data are presented as means ± SEM. NS, not significant ( p > 0.05); * p < 0.05; ** p < 0.01. Statistical significance was calculated using t -tests.

Article Snippet: The following antibodies were used: anti-Janus kinase-1 (JAK1) (bs-1439R, Bioss, Beijing, China), anti-phospho (p)-JAK1 (Tyr1034 + Tyr1035, bs-3238R, Bioss, China), anti- signal transducer and activator of transcription 6 (STAT6) (380957, ZEN BIO, China), anti-p-STAT6 (Tyr641, ab263947, Abcam, Cambridge, MA, USA), anti-pIgR (AF2800-SP, R&D Systems, Minneapolis, MN, USA), anti-TGFβ1 (bs-4538R, Bioss, China), and anti-β-actin (AP0060, Bioworld Technology Inc., Bloomington, MN, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 1: Comparison on the down-regulation of Moutan Cortex from different regions on protein expression levels of ICAM-1,TGF-β1 and FN. HBZY-1 mesangial cells were treated with 200 μg/mL AGEs in the presence or absence of Moutan Cortex (MC) extract of 200 μg/mL. Aminoguanidine of 10 μM was used as the positive control while BSA (200 μg/mL) as blank control. (A) Western blotting was performed to compare the protein expression levels. (B–D) The grayscale scan results of ICAM-1, TGF-β1 and FN. a, Control; b, 200 μg/mL AGEs; c, Positive control aminoguanidine group; “d-m” represent MC from Anhui, Guizhou, Zhejiang, Henan, Hunan, Hebei, Sichuan, Chongqing, Shandong, Gansu. Data are expressed as means ± SD, n = 3. ###P < 0.001 vs. BSA group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. AGEs group; $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. MC from Anhui group.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Comparison, Expressing, Positive Control, Control, Western Blot

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 2: Effect of Moutan Cortex from different regions on ICAM-1 and TGF-β1 protein expression levels in kidney of DN rats. After being treated with STZ and/or MC extract of 5g/kg or , immunohistochemistry was conducted to evaluate the expression levels of ICAM-1 (A) and TGF-β1 (B) of renal tissues. “a” represents normal control; “b” represents model group (DN rats); “c” represents Positive control 0.1 g/kg AG; “d-m” represent Gansu, Chongqing, Shangdong, Sichuan, Zhejiang, Anhui, Hunan, Guizhou, Hebei, Henan.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Control, Positive Control