Journal: International Journal of Molecular Sciences
Article Title: 3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1
doi: 10.3390/ijms241915020
Figure Lengend Snippet: FDFT1 mediated the cytotoxicity of ATA towards HCT116 cells. ( A ) The gene expression levels of FDFT1 in the HCT116 cells treated with ATA for different times via the RT-qPCR assay. ( B , C ) The protein expression levels of FDFT1 in the HCT116 cells treated with ATA (7.5, 15 and 30 μM) for 24 h via Western blotting. The figure ( B ) shown is representative of three independent experiments. The data ( C ) are expressed as means ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. Ctrl (0 μM). ( D – F ) After pre-incubation with or without the FDFT1 inhibitor YM-53601 (2 and 4 µM) for 30 min, HCT116 cells were treated with ATA (0 µM, 5 µM, and 7.5 µM, D), OA (100 μM, E), or UA (40 μM, F) for 24 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 vs. the control (Ctrl). ( G – H ) After pre-incubation with or without YM-53601 (4 µM, 30 min), the HCT116 cells were treated with ATA (20 μM) for 24 h. The autophagic flux was observed through the mRFP-GFP-LC3 assay. The figure ( G ) shown is representative of three independent experiments. The cell apoptosis was determined using FCM. The figure ( H ) is representative of three independent experiments. The apoptotic percentages ( I ) are expressed as means ± SD ( n = 3). *** p < 0.001 vs. Ctrl. ( J – L ) The mRNA ( J ) and protein ( K , L ) expression levels of FDFT1 in HCT116 cells were detected using RT-qPCR and Western blotting after transfection with FDFT1 siRNA for 24 h and 48 h, respectively. siNC—negative control siRNA. The figure ( K ) shown is representative of three independent experiments. The data ( J , L ) are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC. ( M ) The HCT116 cells were transfected with FDFT1 siRNAs for 48 h, followed by exposure to ATA at indicated concentrations for another 48 h. The cell viabilities were detected through the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC.
Article Snippet: For this study, 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, St. Louis, MO, USA; cell culture medium, trypsin, penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Gibco, Burlington, MA, USA; TRIzol was purchased from Invitrogen, Carlsbad, CA, USA; reverse transcriptase, oligo(dT) 18 , and ribonuclease inhibitor were obtained from Shanghai Sangon Biotech Co., Ltd., Shanghai, China; RIPA lysis buffer, BCA protein assay kit, anti-mouse actin mAbs, anti-caspase3 antibody (AF0081), horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (H + L), enhanced chemiluminescence (ECL) kit, and reactive oxygen species (ROS) assay kit were purchased from Beyotime, Shanghai, China; phosphatase inhibitor and protease inhibitor cocktails were obtained from Biotool, Houston, TX, USA; anti-FDFT1 antibody (ab195046) was purchased from Abcam, Cambridge, UK; anti-cyclinD1 antibody (ET1601-31) was obtained from HUABIO, Shanghai, China; anti-GPX4 (67763-1-Ig) antibody was obtained from Proteintech, Rosemont, IL, USA; the antibodies against LC3A/B (4108S) and p62 (39749S) were purchased from CST, Danvers, MA, USA; a monodansylcadaverine (MDC) sensor kit was obtained from KeyGEN BioTECH Corp., Ltd., Nanjing, China; an annexin V-FITC apoptosis detection kit and propidium iodide (PI)/RNase staining buffer were obtained from BD Pharmingen, San Diego, CA, USA; mRFP-GFP-LC3 lentivirus was obtained from Genechem, Shanghai, China.
Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation, MTT Assay, Transfection, Negative Control
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