Histologic, Masson and IHC analyses were performed according to the previously reported methods (Qiao et al., 2020 (link)) with modifications. For histological analysis, lung sections were stained with haematoxylin and eosin (H&E) and Masson. The antibodies against α-SMA (CST, Beverly, MA, United States), TGF-β (Proteintech, United States) were used for IHC staining in lung tissue sections, the percentage of TGF-β and α-SMA positive area (%) from IHC were quantified by ImageJ software. The stained sections were observed and the images were acquired using a microscope (ZEISS AXIO microscope, Axio Scope A1, United States).
Tgf β
Manufactured by Proteintech
Sourced in United States, China
TGF-β is a lab equipment product that functions as a cytokine. It is involved in the regulation of various cellular processes, including cell growth, differentiation, and immune response.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (7 mentions)
α sma, by Proteintech (4 mentions)
Tnf α, by Proteintech (3 mentions)
E cadherin, by Proteintech (3 mentions)
N cadherin, by Proteintech (3 mentions)
Gapdh, by Proteintech (3 mentions)
Cd206, by Proteintech (3 mentions)
Collagen 1, by Proteintech (3 mentions)
Pparγ, by Proteintech (2 mentions)
Vimentin, by Proteintech (2 mentions)
P smad3, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Collagen 3, by Proteintech (2 mentions)
Smad3, by Proteintech (2 mentions)
Smad2, by Proteintech (2 mentions)
Anti col4, by Abcam (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
24 protocols using tgf β
1
Histological and IHC Analysis of Lung Tissue
2
Evaluation of Fibrosis-related Proteins
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Anti-GAPDH, anti-Hsp90, anti-β-actin, anti-Col І, anti-Col IV, anti-vimentin, anti-α-SMA, anti-fibronectin, and anti-CTGF antibodies were obtained from Abcam (Cambridge Science Park, Cambridge, UK). Anti-p53, p-Smad3, and Smad3 were purchased from Cell Signaling Technology (Danvers, MA, USA), while anti-COXIV and TGF-β were obtained from Proteintech (Rosemont, IL, USA). Anti-DsbA-L antibody was provided by Dr. Feng Liu Lab. All secondary antibodies, MitoTracker Green FM and MitoTracker Red CMXRos, were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). The cisplatin, aristolochic acid, and Mitochondria Isolation Kit were purchased from Sigma-Aldrich (Shanghai, China). The target sequence for mouse Hsp90β was described as in the previous study40 (link).
3
Molecular Signaling in Tissue Repair
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Primary antibodies against p38 MAPK (Cat. No. ab31828), collagen I (Cat. No. ab34710), collagen III (Cat. No. ab7778), fibronectin (Cat. No.ab2413), HBEGF (Cat. No. ab92620), and KRAS (Cat. no. ab180772) were purchased from Abcam (Cambridge, MA, USA). Anti- GAPDH antibody (Cat. No. 10494-1-AP) and TGF-β were obtained from Proteintech North America (Rosemont, IL, USA). Anti-phospho-p38MAPK antibody (Cat. No. 4511) was purchased from Cell Signaling Technology (Danvers, MA, USA). The luciferase assay kit was purchased from BioVision (Milpitas, CA, USA). All plasmids used in this study were generated by Vigene Biosciences (Jinan, Shangdong, China). The p38 MAPK inhibitor, SB203580 (Cat. No. HY-10256) and p38MAPK agonist (Cat. #HY-N0674A/CS-6061) were purchased from MedChemExpress USA (Deer Park, NJ, USA).
4
Signaling Pathway Protein Analysis
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Western blot analysis was conducted as previously described.20 (link) Antibodies for GFAP, intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β) were obtained from Proteintech (Chicago, IL, USA). Antibodies for VEGF, TGF-β receptor II (TGF-βRII), Smad2/3, and fibronectin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for alpha smooth muscle actin (α-SMA) and β-actin were obtained from Sigma-Aldrich. An antibody for phospho-Smad2/3 (P-Smad2/3) was purchased from Cell Signaling (Danvers, MA, USA).
5
Comprehensive Cardiac Protein Analysis
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Heart tissues or cultured cells were sonicated in RIPA lysis buffer and homogenized. The debris was removed and the supernatant was obtained after centrifugation at 12,000 g for 10 min at 4°C. About 30–50 μg proteins was loaded for electrophoresis, and probed with primary antibodies against collagen I (1:1000; No.14695-1-AP; Proteintech Co., Wuhan, China), α-SMA (1:1000; No.14395-1-AP; Proteintech Co.), TGF-β (1:1000; No.21898-1-AP; Proteintech Co.), LC3 I/II (1:1000; No.14600-1-AP; Proteintech Co., Wuhan, China), TIM23 (1:1000; No.11123-1-AP; Proteintech Co., Wuhan, China), TOM20 (1:1000; No.66777-1-Ig; Proteintech Co., Wuhan, China), OPTN (1:1000; No.10837-1-AP; Proteintech Co., Wuhan, China), COX4 (1:1000; No.11242-1-AP; Proteintech Co., Wuhan, China). GAPDH (1:1000, AF0006; Beyotime Biotechnology Co., Shanghai, China) was used as internal control. Images were analyzed using the Image-Pro Plus software.
6
Chitosan-Stearic Acid Nanoparticle Synthesis
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Chitosan (Mw=450kDa, Yuhuan, China). The stearic acid (SA) was purchased from Shanghai Chemical Reagent Co., Ltd. Both 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) were purchased from Shanghai Medpep Co, Ltd. Doxorubicin hydrochlorate (DOX.HCl) was obtained from Dalian Meilun Biotech Co., Ltd. Telmisartan and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were acquired from Sigma-Aldrich Inc. (St Louis, MO). Indocyanine green (ICG) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Trypsin and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco-BRL Life Technologies (Carlsbad, CA). Fetal calf serum was purchased from Sijiqing Biology Engineering Materials Co, Ltd (Zhejiang, China). Primary antibodies for α-SMA, TGF-β, CD31, CCL2, PPAR-γ, C-Caspase, HIF-α and HABP were purchased from Proteintech Group, Inc. CCL2 primary antibodies was purchased from Wuhan Servicebio technology co., LTD. Other chemicals used were of chromatographic grade or analytical grade.
7
Ginsenoside Rh4 Anticancer Properties
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Ginsenoside Rh4 (Supplementary Figure S1A , purity ≥ 99%) was purchased from Puruifa Technology Development Co., Ltd. (Chengdu, China). Methylthiazoletetrazolium (MTT) and dimethyl sulfoxide (DMSO) were obtained from Aladdin Biotechnology (Shanghai, China). Fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Gibco (Grand Island, NY, USA). Dulbecco’s modified Eagle medium (DMEM) was purchased from HyClone (Logan, UT, USA). The 24-well Transwell chambers were obtained from Corning (New York, NY, USA). Lipofectamine 2000 and TRIzol Reagent were supplied by Invitrogen (Carlsbad, CA, USA). SYBR Green Master Mixture was obtained from Takara (Otsu, Japan). Primary antibodies against E-cadherin, Vimentin, N-cadherin, Snail1, SIX1, and TGF-β were purchased from Proteintech Group Inc. (Chicago, IL, USA). The primary antibodies against Smad3, P-Smad3, and β-actin were supplied by Abcam (Cambridge UK). Goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Abbkine (Wuhan, China). Oxaliplatin was obtained from Qilu Pharmaceutical Co., Ltd. (Shandong, China).
8
Liver Tissue Protein Expression Analysis
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Liver tissues were homogenized and determined as described previously (Wang et al., 2019 (link)). The blots were incubated with primary antibodies: cleaved caspase-3, Bax, Bcl-2, ATF6, GRP78, Nrf2, SOD2, collagen IA1, ATG5, p62 and LC3 (Abcam, USA), HO-1 (Santa Cruz Biotechnology, CA, USA), CHOP, P-IRE1α, P-eIF2α, P-PERK, PERK, FGFR1, P-AMPK and AMPK (Cell Signaling Technology, Danvers, MA, US), and α-SMA, TGF-β, and GAPDH (Proteintech, China) followed by incubation with their corresponding secondary antibodies: anti-rabbit and anti-mouse (Proteintech, China).
9
HUVEC Culture and Manipulation
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Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in Endothelial Cell Basal Medium-2 (Lonza, CC-3156) added with EGM™-2 SingleQuots™ Supplements (Lonza, CC-4176). HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (HyClone, SH30023.01) supplemented with 10% FBS. HUVECs were treated with NaHS (0.1, 1 mM), verteporfin (0.25 μM), or the control vehicle for 24 h. HUVECs were treated with NaHS (1 mM) for 24 h for qRT-PCR, or treated with TGFβ (Proteintech; 20 ng/mL) for 24 h for RNA-seq. For lentivirus construction, the targeting shRNAs (shPRKAA1, 5′-GTTGCCTACCATCTCATAATA-3’; shYAP, 5-CCCAGTTAAATGTTCACCAAT-3′) were cloned into PLKO.1 plasmid and generated in HEK293T first. Then, HUVECs were infected with lentivirus carrying shPRKAA1, shYAP, or negative control. Puromycin (1 μg/mL) was used to screen successfully infected cells after 48 h. For knockdown of MPST by siRNA, siMPST (siMPST-1#, 5′-AGAGTGTTTCTTCACTCAA-3’; siMPST-2#, 5′-AGACCTACGAGGACATCAA-3′) or negative control were transfected into HUVECs with Lipofectamine RNAiMAX (Invitrogen, 13778150) for 48 h.
10
Western Blotting Analysis of Protein Markers
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