Cell counting kit 8 ck04

Antibodies used in this study were principally purchased from Santa Cruz Biotechnology (G6PD (G-12): SC-373886 (dilution 1:1000), 6PGD (G-2): SC-398977 (dilution 1:1000), His (HIS.H8): SC-57598 (dilution 1:1000), Enolase (H-300): SC-15343 (dilution 1:1000)), Cell Signaling Technology (beta actin (8H10D10): 3700 S (dilution 1:1000)), Everest Biotech (G6PD: EB07841 (dilution 1:1000)), Advance Immunochemical (GAPDH (6C5): 6C5 (dilution 1:2000)), and Abcam (G6PD: AB87230 (dilution 1:1000), band 3 (EPR1426): AB108414 (dilution 1:1000)). TALON Superflow (28-9575-02) and bovine thrombin (91-030) were purchased from GE Healthcare and BioPharma for protein purification. Pfu Turbo polymerase (600252) used for site-directed mutagenesis was purchased from Agilent Technologies. Chymotrypsin was purchased from Promega (V1061). Cell counting kit-8 (CK04) and glutathione quantification kit (T419) were purchased from Dojindo. Cycloheximide (C7698) and chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA, C6827) were purchased from Sigma and Thermo Fisher Scientific, respectively. Glutathione assay kit (703002) used for blood assays was purchased from Cayman Chemical.

Forty-eight hours after siRNA transfection, cells were collected and plated in 96-well plates at a density of 2,000 cells per well. A CCK8 (Cell Counting Kit-8, CK04, Dojindo) assay was used to analyze cell proliferation on days 0, 1, 2, 3, and 4. After 2 hours incubation with CCK8 reagent, the number of viable cells was recorded by measuring the absorbance at 450 nm.

BLM (B107423) and OST (O101699) were purchased from Aladdin (Shanghai, China), and OST was isolated from the fruit of Cnidium ninidium with purity of 99%. TGF-β1 (H8541) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against vimentin (ab8978), collagen I (ab34710), collagen III (ab7778), connective tissue growth factor (CTGF, ab6992), 4HNE (ab48506), p-Smad3 (ab52903), and t-Smad3 (ab40854) were purchased from Abcam (Cambridge, MA, USA). Antibodies against α-smooth muscle actin (α-SMA, A2547, for Western blot analysis) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against TGF-β (21898-1-AP), NOX4 (14347-1-AP), α-SMA (14395-1-AP, for immunofluorescence staining), and β-actin (60008-1-Ig) were purchased from Proteintech (Hubei, China). The reactive oxygen species (ROS) kit (S0033) was purchased from Beyotime (Shanghai, China), cell counting kit-8 (CCK8) (CK04) was purchased from Dojindo (Shanghai, China), and the Cytotoxicity Detection Kit (11644793001) was purchased from Roche (Hoffmann, Germany). The hydroxyproline assay kit (Cat No.A030-2) was purchased from Jiancheng Co. (Jiangsu, China), and the MDA assay kit (Cat No.S0131) was purchased from Beyotime Co. (Shanghai, China).

Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12, 11330), fetal bovine serum (FBS, 12676), and 0.25% trypsin (25200-056) were purchased from Life Technologies (NY, USA). Testosterone was a kind gift from Beijing Zizhu Pharmaceutical Company (Beijing, China). A cell counting kit-8 (CCK-8, CK04) was purchased from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). Fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit and propidium iodide (PI)/RNase staining buffer (550825) were purchased from BD Biosciences (CA, USA). An In Situ Cell Death Detection Kit (fluorescein, 11684795910) was purchased from Roche (Mannheim, Germany). Rabbit anti-rat β-actin (ab8227), cytochrome c (ab53056), caspase-3 (ab47131), caspase-8 (ab25901), and caspase-9 polyclonal antibodies (ab2013) were purchased from Abcam Inc. (Cambridge, MA, USA). Prestained protein ladder (26616) and Halt protease inhibitor cocktail (186229) were purchased from Thermo Scientific (IL, USA). A tumor necrosis factor alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA) kit (RTA00) was purchased from R&D Systems Inc. (MN, USA). All other chemicals were purchased from either Sigma-Aldrich (MO, USA) or Beyotime Institute of Biotechnology (Nantong, Jiangsu, China).