Ab32147

Human ARPE-19 cell line was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Phillyrin (batch number Z17A8X34077, purity > 98%) was a product of the China Food and Drug Administration (Beijing, China). Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, and streptomycin solutions were purchased from Corning (NY, USA). Trypsin (0.05%) and phosphate buffered saline (PBS) were produced by Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT), ML385, N-acetylcysteine (NAC)m and H₂O₂ solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Fas (ab82419), cytochrome c (ab90529), cleaved caspase-3 (ab2302), cleaved caspase-9 (ab2324), NQO1 (ab80588), Keap1 (ab118285), Bcl-2 (ab185002), Nrf2 (ab62352), CDK2 (ab32147), cyclin A (ab33911), cyclin E (ab181591), Bax (ab53154), β-actin (ab8226), p21 (ab188224), Histone H3 (ab1791), COX IV (ab16056) p53 (ab241556), pro caspase-3, pro caspase-8, and pro caspase-9 were obtained from Abcam, Cambridge, (MA, USA). Antibodies against p-p53 (9286S), cleaved caspase-8 (9496S), cleaved PARP (5625S), and HO-1 (86806S) were purchased from Cell Signaling Technology, Beverly, (MA, USA).

The following antibodies were purchased from Abcam: OLA1 (ab229090), GAPDH (ab8245), CCNB1 (ab71977), CCNB2 (ab18250), CCNE2 (ab40890), CDK2 (ab32147), CDK4 (ab108357), P21 (ab188224), Bcl-2 (ab32124), BAD (ab32445), cleaved caspase 3 (ab32042), Rb (ab181616), pRb (ab184796), and E2F1 (ab112580). The P21 inhibitor UC2288 was purchased from Abcam (ab146969) and used as recommended.

Total protein in tissues or cells was extracted with radio immunoprecipitation assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride (P0013C; Beyotime Institute of Biotechnology), with the concentration detected by BCA kit. After sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the protein was electrotransferred onto a polyvinylidene fluoride membrane and blocked with 5% skim milk powder at room temperature for 1 hour. Then, the membrane was probed with diluted primary antibodies to GSK3β (1:5000, ab32391; Abcam), FTO (1:1500, ab126605; Abcam), MZF1 (1:500, ab64866; Abcam,), c‐Myc (1:1000, ab32072; Abcam), CDK2 (1:1000, ab32147; Abcam), CDK4 (1:500, ab137675; Abcam), Ki‐67 (1:1000, ab16667; Abcam), PCNA (1:1000, ab18197; Abcam), Bax (1:1000, ab199677; Abcam), Bcl‐2 (1:500, ab59348; Abcam) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (ab9485, 1:2500; Abcam) at 4°C overnight. Next day, the membrane was re‐probed with horseradish peroxidase (HRP)‐labelled secondary antibody of goat anti‐rabbit antibody to immunoglobulin G (IgG) for 1 hour and visualized by enhanced chemiluminescence (ECL) kit (BB‐3501, Ameshame; Chiltem). Finally, Quantity One v4.6.2 software was used to quantify the grey levels of each band in the Western blot image. GAPDH served as an internal reference.

The RIPA lysis buffer was purchased from Solarbio (Beijing, China) to lyse the NSCLC cells/tissues and extract the total protein, according to the experimental procedures recorded in the previous publications [36 (link), 37 (link)], the expression levels of GOT1, β-actin, cyclin D1, CDK2, cleaved caspase-3 and Bax were examined by using the Western Blot analysis. Specifically, the 40 μg/lane protein lysates were separated by using the 10 -15% SDS-PAGE, and the target protein bands were transferred onto the PVDF membranes (Millipore, USA). Next, the membranes were incubated with 5% skim milk for 70 min at room temperature for blocking, and the membranes were probed with the primary antibodies against GOT1 (1:1500, MW: 50 kDa, #PA5–24634, Thermo, USA), β-actin (1:2000, MW: 42 kDa, #ab6276, Abcam, UK), cyclin D1 (1:1500, MW: 35 kDa, #ab40754, Abcam, UK), CDK2 (1:2000, MW: 33 kDa, #ab32147, Abcam, UK), cleaved caspase-3 (1:1500, MW: 17 kDa, #ab32042, Abcam, UK) and Bax (1:1500, MW: 21 kDa, #ab32503, Abcam, UK) overnight at 4 °C. After washing by PBS buffer for 3 times, the PVDF membranes were incubated with the secondary antibody (Abcam, UK) for 120 min at room temperature. Finally, the protein bands were visualized by ECL system (GE Healthcare Bio-science, USA) and quantified by using the Image J software.

Proteins were extracted in RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail and a phosphatase inhibitor (Bimake, USA) and determined by the BCA assay kit (Thermo, USA). After polyacrylamide gel electrophoresis, proteins were transferred to a PVDF membrane and incubated overnight at 4°C with specific primary antibodies. Antibodies against PARK2 (ab15954), CDK2 (ab32147), mTOR (ab2732), p-mTOR (ab109268), EGFR (ab52894), p-EGFR (ab40815), and AKT (ab8805) were purchased from Abcam (Cambridge, MA, USA). E-cadherin (3195), N-cadherin (13116), vimentin (5741), slug (9585) claudin-1 (13255), cyclin D1 (2978), CDK4 (12790), cyclin D3 (2936), P21 (2947), P18 (2896), cleaved caspase-3 (9664), cleaved caspase-8 (8592), cleaved caspase-9 (20750), cleaved PARP (5625), Cytochrome c (11940), p-AKT (4060), and GAPDH (2118) were obtained from Cell Signaling Technology (Boston, USA). The goat anti-rabbit IgG–HRP (HA1001-100) and goat anti-mouse IgG–HRP (HA1006) were obtained from HuaAn Biotechnology (Huabio, China). Then the PVDF membrane was incubated with secondary antibody at room temperature for 1 h. The membranes were observed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo, USA). Band intensity was analyzed with Quantity One.