Naïve CD4 T cells were cultured in precoated plates with anti‐CD3 (5 μg/mL, Clone OKT‐3, BioLegend, USA) + anti‐CD28 (1 μg/mL, Clone CD28.2，BioLegend, USA) antibodies and maintained in media supplemented with IL‐2 (10 ng/mL, PeproTech, USA) (Group Th0). Recombinant IL‐6 (50 ng/mL, PeproTech, USA), recombinant TGF‐β1 (10 ng/mL, PeproTech, USA), anti‐IFN‐γ mAb (10 μg/mL, Clone B27, BioLegend, USA), and anti‐IL‐4 mAb (10 μg/mL, Clone 8D4‐8, BioLegend, USA) were added to generate Th17 cells (Group Th17). recombinant TGF‐β1 (50 ng/mL, PeproTech, USA), anti‐IL‐6 mAb (10 μg/mL, Clone MQ2‐13A5, BioLegend, USA), anti‐IFN‐γ mAb (10 μg/mL, Clone B27, BioLegend, USA), and anti‐IL‐4 mAb (10 μg/mL, Clone 8D4‐8, BioLegend, USA) were added to generate Treg cells (Group Treg). Media were changed every 48 hours. This protocol was based on previous publications.17 , 18 For intracellular cytokine analysis, brefeldin A (10 mg/mL, BioLegend, USA), phorbol 12‐myrstate 13‐acetate (PMA)(50 ng/mL, BioLegend, USA), and ionomycin (1 μg/mL, BioLegend, USA) were added 4 hours before the end of the culture. The cells were then harvested, stained, and analyzed through flow cytometry. After 5 days of culture, 8.3 ± 1.7% of CD4+IL‐17A+ cells and 13.2 ± 1.7% of CD4+Foxp3+ cells were obtained.
Recombinant tgf β1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant TGF-β1 is a cytokine protein produced using recombinant DNA technology. It is a member of the transforming growth factor beta family and plays a role in cellular processes such as cell growth, cell differentiation, and immune function.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Leupeptin, by Merck Group (2 mentions)
Phospho smad3, by Cell Signaling Technology (2 mentions)
Tβrii, by Santa Cruz Biotechnology (2 mentions)
Phospho smad2, by Cell Signaling Technology (2 mentions)
Recombinant il 1β, by Thermo Fisher Scientific (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Rpmi 1640 medium, by American Type Culture Collection (2 mentions)
Recombinant tnf α, by R&D Systems (2 mentions)
Recombinant il 17, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Sb431542, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Soluble anti cd28, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
43 protocols using recombinant tgf β1
1
Th17 and Treg Cell Differentiation Protocol
2
Fibroblast Cell Culture and TGF-β Signaling
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Human fetal lung fibroblast (MRC5) cell lines were propagated in EMEM media (Gibco, Rockville, IL, USA) with 10% FBS (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin antibiotic mix (Lonza, Allendale, NJ, USA). The cells were kept in a 37 °C humidified incubator with 5% carbon dioxide. Immobilized protein A/G beads, FN, α-SMA, TβRI, HA tag, USP11, V5 tag, TβRII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SMAD2, phospho-SMAD3, total SMAD2, SMAD3, and ubiquitin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bleomycin, leupeptin, CHX, MTX, and antibodies against Flag-tag and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG-132 was from Calbiochem (KGaA, Darmstadt, Germany). DAPI was purchased from ThermoFisher Scientific (Waltham, MA, USA). All materials in highest grades used in the experiments are commercially available.
3
Isolation and Stimulation of Colonic LPMCs
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LPMCs were isolated from colon tissue of a control WT mouse based on the method described by McManus et al. [29 (link)]. Briefly, the total colon was harvested and cleaned thoroughly by PBS, thereafter cut into small pieces. Tissue fragments were digested in collagenase containing solution (1 mg/ml; Sigma-Aldrich), then mechanically disaggregated and filtered using Falcon 40 μm cell strainer (Thermo Fisher Scientific, Waltham, MA, USA) to obtain single-cell suspension. LPMCs were then isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). After isolation, cells were placed into RPMI 1640 medium (ATCC) supplemented with L-glutamine, 10% FBS and 1% streptomycin and penicillin mixture. To perform PCRs LPMCs were seeded into 96-well plates at a density of 5 × 105 cells/well (n = 6 well/treatment group) and treated either with recombinant IL-1β (100 ng/ml; Invitrogen), LPS (100 ng/ml; Lipopolysaccharides from Escherichia coli, Sigma-Aldrich), recombinant TNF-α (10 ng/ml; R&D), recombinant TGF-β1 (0.5 nM; Invitrogen), recombinant IL-17 (100 ng/ml; R&D), or 25 µM H2O2 for 24 h. Vehicle treated cells served as controls.
4
siRNA Knockdown of EMT Regulators in ESCC Cell Lines
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We used TE5, TE6, TE8, and TE11 human cell lines derived from poorly, well, moderately, and moderately ESCC, respectively. Cells were maintained in RPMI‐1640 medium plus 20% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37°C. TE5, TE6 and TE11 cell lines had epithelial characteristics and the TE8 cell line had EMT characteristics.
For siRNA transfections, TE8 cells were seeded in 6‐well plate (0.25 × 106 cells per well) and transfected with 30 pmol of siRNA using Lipofectamine RNAimax reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 48 h, according to the procedure provided by the manufacturer. The siRNAs specific for ZEB1 were Stealth siRNAs named HSS110548 and HSS186235 (referred as ZEB1‐1 and ZEB1‐2, respectively) (Thermo Fisher Scientific). The siRNAs specific for Twist and Snail were HSS144372 and HSS186975 (referred as Twist‐1 and Twist‐2, respectively) and HSS143995 and HSS143996 (referred as Snail‐1 and Snail‐2, respectively). Stealth RNAi siRNA negative control duplex was used as nontargeting (NT) siRNA. In TE8 cells, IFN‐γ (R&D Systems, Minneapolis, MN, USA) was used at 5 ng/mL.
For TGF‐β1 treatment, TE5, TE6, and TE11 cells were seeded in a 6‐well plate (0.25 × 106 cells per well) and cells were treated for 96 h with recombinant TGF‐β1 (Invitrogen, Carlsbad, CA, USA) at a final concentration of 20 ng/mL.
For siRNA transfections, TE8 cells were seeded in 6‐well plate (0.25 × 106 cells per well) and transfected with 30 pmol of siRNA using Lipofectamine RNAimax reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 48 h, according to the procedure provided by the manufacturer. The siRNAs specific for ZEB1 were Stealth siRNAs named HSS110548 and HSS186235 (referred as ZEB1‐1 and ZEB1‐2, respectively) (Thermo Fisher Scientific). The siRNAs specific for Twist and Snail were HSS144372 and HSS186975 (referred as Twist‐1 and Twist‐2, respectively) and HSS143995 and HSS143996 (referred as Snail‐1 and Snail‐2, respectively). Stealth RNAi siRNA negative control duplex was used as nontargeting (NT) siRNA. In TE8 cells, IFN‐γ (R&D Systems, Minneapolis, MN, USA) was used at 5 ng/mL.
For TGF‐β1 treatment, TE5, TE6, and TE11 cells were seeded in a 6‐well plate (0.25 × 106 cells per well) and cells were treated for 96 h with recombinant TGF‐β1 (Invitrogen, Carlsbad, CA, USA) at a final concentration of 20 ng/mL.
5
Isolation and Stimulation of Pediatric PBMCs
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PBMCs from control pediatric patient (5 yearsoldmale, gastroenterological examination due to tongue plaque andhalitosis) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). After isolation, cells were placed into RPMI 1640 medium (ATCC) supplemented with L-glutamine, 10% FBS and 1% streptomycin and penicillin mixture. To perform mRNA expression analysis, PBMCs were seeded into 24-well plates at a density of 5 × 105 cells/well (n = 6 well/treatment group) and treated either with recombinant IL-1β (100 ng/ml; Invitrogen), LPS (100 ng/ml; Lipopolysaccharides from Escherichia coli, Sigma-Aldrich), recombinant TNF-α (10 ng/ml; R&D), recombinant TGF-β1 (0.5 nM; Invitrogen), recombinant IL-17 (100 ng/ml; R&D), or 25 µM H2O2 for 24 h. Vehicle treated cells served as controls.
6
Cell Culture Techniques for Esophageal, Kidney, and Lung Cancer
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Esophageal adenocarcinoma (OE19 and OE33) cancer cells [Sigma-Aldrich (St. Louis, MO, USA)] were cultured with RPMI 1640 medium containing 2 mM glutamine and 10% FBS. HEK293 cells [Invitrogen (Carlsbad, CA, USA)] were cultured with DMEM medium containing 10% FBS. Murine lung epithelia (MLE12) cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured with HITES medium containing 10% FBS. All the cells were cultured at 37°C in 5% CO2. V5 antibody, E-cadherine antibody, mammalian expressional plasmid pcDNA3.1D/His V5 TOPO, Escherichia coli Top 10 competent cells, and recombinant TGFβ1 were from Invitrogen (Carlsbad, CA, USA). HA tag (29 F4), Flag tag (9A3), and ubiquitin (P4D1) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Rac3 antibody was from Proteintech Group (Chicago, IL, USA). FBXL19 antibody was from Abgent (San Diego, CA, USA). Leupeptin, ammonium chloride (NH4Cl), β-actin antibody, individual FBXL19 shRNAs, and scrambled shRNA were from Sigma Aldrich (St. Louis, MO, USA). MG132 and lactacystin from Calbiochem (La Jolla, CA, USA). Immunobilized protein A/G beads were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Superfect transfection reagent was from QIAGEN (Valencia, CA, USA). All materials in highest grades uses in the experiments are commercially available.
7
Mouse AdMSCs Isolation and TGF-β1/Rosiglitazone Treatment
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Mouse AdMSCs were isolated from adult male C57BL/6J mice’s inguinal fat pads as previously described (28 (link)). Briefly, the inguinal fat pads from 8- to 10-week-old male C57BL/6 mice were harvested, washed with PBS, and minced, followed by digestion in 10 mL of type I collagenase (1 mg/mL in 1% BSA/PBS) for 30 minutes at 37°C. The digested fat pads were filtered through a 40 μm cell strainer and centrifuged at 500g for 5 minutes at room temperature. The supernatant containing adipocytes and debris was discarded. Pelleted cells were resuspended and washed with PBS 2 times and used as AdMSCs. AdMSCs were cultured in DMEM/F12 (MilliporeSigma) supplemented with 10% fetal bovine serum (MilliporeSigma) and antibiotics (penicillin and streptomycin, Hyclone). AdMSCs were maintained at 37°C under a 5% CO2 atmosphere. For the rosiglitazone treatment experiment, AdMSCs were treated with/without recombinant TGF-β1 (10 ng/mL) (Invitrogen) and rosiglitazone (10 μM) for 7 days after seeding (10 × 104 cells/mL) in 6-well plates.
8
DDR1 Inhibitor 7rh Synthesis and Signaling
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DDR1 inhibitor 7rh (chemical structure, Fig. 1i ) was synthesized in our laboratory.12 (link) TGFβRI inhibitor SB525334 was obtained from Selleck (Shanghai, China). Annexin V-FITC and collagen I were from Sigma-Aldrich (Shanghai, China). Recombinant TGF-β1 was purchased from Invitrogen (Shanghai, China). Antibodies against DDR1, Survivin, and Bcl-XL were from Santa Cruz Biotech (Santa Cruz, CA). Antibodies against PARP (clone 4C10-5), caspase-3, XIAP, and Bcl-2 were purchased from BD Biosciences (San Jose, CA). Antibodies against DDR2, STAT3, p-STAT3 (Y705), p-DDR1 (Y513), active Caspase-3, Bax, α-SMA, SOX2, NANOG, OCT4, KLF4, SLUG and ALDH were from Cell Signaling Technology (Beverly, MA). Anti-β-actin was from Sigma-Aldrich (Shanghai, China). Secondary antibodies used were fluorescent-conjugated anti-mouse IgG and anti-rabbit IgG (LI-COR Biotechnology, Nebraska, USA).
9
TGF-β1 Induced Fibrosis in MRC-5 Lung Fibroblasts
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Human embryonic lung fibroblasts (MRC-5) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). For maintenance, the cells (MRC-5) were maintained in the appropriate medium [Dulbeccos Modified Eagle Medium (DMEM)] containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco) at 37 ℃ with 5% carbon dioxide (CO2). Before determining the concentration, we searched lots of literature and found many studies (29 (link)-31 (link)) finally determined 5 ng/mL TGF-β1 as the optimal concentration by setting different concentration and comparing the effect of promoting fibrosis. In the pre-experiment we also set different concentration of TGF-β1 (2, 5, 10 ng/mL), and found when the concentration of TGF-β1 is 5 ng/mL, the effect of promoting fibrosis is the most significant. Therefore, fibroblasts (MRC-5) were cultured in a medium supplemented with 5 ng/mL recombinant TGF-β1 (Peprotech, New Jersey, USA) for 0, 24, and 48 h to induce fibroblast activation.
10
Antifibrotic Effects of α-MG in Lung Fibrosis
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α-MG (purity over 99%) was prepared by Chengdu Biopurify Phytochemical Ltd. (Chengdu, China). BLM was obtained from Nippon Kayaku (Tokyo, Japan). NDN (40 mg/kg) was obtained from Shanghai Yuke Chemical Co., Ltd. (Shanghai, China). Recombinant TGF-β1 was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). Compound C (CC) was provided by Selleck (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Biosharp Technology Inc. (Hefei, China).
A Hydroxyproline (HYP) Assay Kit was purchased from Nanjing Jiancheng Bio-Engineering Institute (Nanjing, China). Antibodies against AMPK, phospho-AMPK (p-AMPK), TGF-β1, Smad2/3, and phospho-Smad2/3 (p-Smad2/3) were purchased from Cell Signal Technology Inc. (Massachusetts, USA). Antibodies against β-actin, alpha-smooth muscle actin (α-SMA), and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology Inc. (Dublin, OH, USA). An antibody against NOX4 was purchased from Abcam Technology Inc. (Cambridge, UK). An antibody against collagen-1 (Col I) was purchased from Wanleibio Technology Inc.
A Hydroxyproline (HYP) Assay Kit was purchased from Nanjing Jiancheng Bio-Engineering Institute (Nanjing, China). Antibodies against AMPK, phospho-AMPK (p-AMPK), TGF-β1, Smad2/3, and phospho-Smad2/3 (p-Smad2/3) were purchased from Cell Signal Technology Inc. (Massachusetts, USA). Antibodies against β-actin, alpha-smooth muscle actin (α-SMA), and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology Inc. (Dublin, OH, USA). An antibody against NOX4 was purchased from Abcam Technology Inc. (Cambridge, UK). An antibody against collagen-1 (Col I) was purchased from Wanleibio Technology Inc.
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