Pifithrin α pft α

MH7A cells were treated with DMEM for 24 h, then with DMEM and 10^–6, 10^–7, 5 × 10^–8, 10^–8 or 10^–9 M VD (Sigma-Aldrich) for 48 h.
MH7A cells were treated with DMEM for 24 h, then with DMEM and 5, 10, 20, 30 or 40 ng/ml TNF-α (Novoprotein Scientific Inc., Shanghai, China) treatment for 48 h. MH7A cells were treated with DMEM for 24 h, then with DMEM and 5, 10, 20, 30 or 40 μM pifithrin-α (PFT-α) (Sigma-Aldrich) (a p53 pro-apoptotic inhibitor) for 48 h.
Normal FLSs were treated with DMEM for 72 h or DMEM and 10^–7 M VD and/or 10 or 30 ng/ml TNF-α (Sigma-Aldrich) for 24 h.

After being subjected to the injection of ginkgetin (25, 50 and 100 mg/kg) or 1% dimethyl sulfoxide in normal saline (NS) (vehicle control, Sigma, St. Louis, MO, U.S.A.) at the onset of reperfusion, the rats (six rats in each group) got injection of 3-methyladenine (3-MA) (Sigma, St. Louis, MO, U.S.A.; 600 nmol) or Pifithrin-α (PFT-α )(Sigma, St. Louis, MO, U.S.A.; 60 nmol) 20 min before ischemia’s beginning to determine the dose-related effects of ginkgetin (Nanjing Puyi Biological Technology Co., Ltd.) (chemical structure shown in Figure 1A) on LC3, histological characteristics and the neurological deficits, or received injection of SN50 (Biomol, Plymouth, PA, U.S.A.; 30 μg) or PFT-α (60 nmol) to reveal the effects of ginkgetin on p53. There were also administration of PFT-a (60 nmol) or SN50 (30 μg) given to rats to elucidate the effects of ginkgetin on DRAM, PUMA, cathepsin B, cathepsin D, Beclin 1, Bcl-2 and Bax.
The protocol of experiment was elucidated in Figure 1B.

The human non-small cell lung cancer cell line A549, HCC827 and H1975 were obtained from American Type Culture Collection (MD, USA). And human normal cell line of MRC-5, L02 and HK2 were purchased from the KeyGEN BioTECH (Nanjing, China). The human NSCLC cell lines A549, HCC827 and H1975 as well as L02 cells were cultured in RPMI 1640 (HyClone, UT, USA) containing 10% (v/v) fetal bovine serum (HyClone), and 1% penicillin/stretomycin (Invitrogen, CA, USA). MRC-5 and HK2 cells were grown in DMEM medium supplemented with (v/v) 10% heat-inactivated FBS and 1% penicillin/stretomycin (Invitrogen). Then, all cells were cultured at 37°C in a humidified atmosphere of 5% CO₂. Juglanin (CAS: 5041-67-8, HPLC≥98%), isolated from leaves of English walnut (Juglans regia), was purchased from Shanghai ZeYe Biological Technology Co., LTD. (Shnghai, China). P53 inhibitor Pifithrin-α (PFT-α, Sigma-Aldrich, MO, USA), NF-κB inhibitor pyrollidine dithiocarbamate (PDTC, Sigma-Aldrich), PI3K inhibitor LY294002 (Selleck Chemicals, TEX, USA), p38 inhibitor p79350 (Beyotime, Jiangsu, China), ERK1/2 inhibitor PD98059 (Selleck Chemicals) and c-Jun N-terminal kinases (JNK) activator Anisomycin (Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (DMSO, KeyGEN BioTECH) and freshly prepared for use.

Prostaglandin A₂ was obtained from Biomol International Inc. (Plymouth Meeting, PA, USA). Pifithrin-α (PFT-α), cycloheximide (CHX) and z-VAD-Fmk were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). NU7441 was from Tocris Bioscience (Bristol, UK). All reagents used in this study were of molecular biology, or cell culture tested grade.

Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA). The cyclin D1, p21, p53, FOXO3a and phosphor-form p53 antibodies were abstained from Epitomics (Burlingame, CA, USA). PD98059 (a special inhibitor of ERK1/2), SB203580 (a special inhibitor of p38 MAPK) were purchased from Merck Millipore (Darmstadt, Germany), MTT powder and Pifithrin-α (PFT-α) were purchased from Sigma Aldrich (St. Louis, MO, USA). p38 MAPK isoforms α and β, p53 and FOXO3a small interfering RNAs (siRNAs) were obtained from Santa Cruz (California, CA, USA). Lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). The FOXO3a-GFP and N1-GFP plasmids were kindly provided by Dr. Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, University Medical Center, Utrecht, the Netherland) and was reported previously [18 (link)]. Berberine was purchased from Chengdu Must Biotechnology (Chengdu, China).

Lactate concentration was measured using the Lactate Colorimetric Assay Kit (Abcam, Cambridge, MA, USA). Equal number of cells were suspend in Lactate Assay Buffer and all experimental procedure was performed according to the instruction of the manufacturer. Experiment was repeated three times and absorbance was measured using the VERSA microplate reader (Turner Biosystems). For p53 inhibition, 20 μM pifithrin-α (PFT-α) (Sigma-Aldrich, MO, USA) was treated for 1 h before the cisplatin treatment and quantifications were made at indicated time points. For N-acetylcysteine (NAC) (Sigma-Aldrich), which is a powerful antioxidant, 10 μM NAC was pretreated for 1 h and then cisplatin was applied. All experiments related to PFT-α and NAC followed the same treatment process.