Cisplatin p4394

The plenti6-SIRT3 expression vector was cloned as previously reported [17 (link)]. The pCMV6-XL5-GSTP1 expression vector (SC119655) was obtained from Origene (USA). Anti-SIRT3 (#2627), Anti-PARP (#9542), Anti-GSTP1 (#3369), Anti-c-Jun (#9165), Anti-phospho-c-Jun (#2361), Anti-phospho-JNK (#9251), Anti-Bim (#2933), Anti-Bax (#5023), Anti-Caspase 9(#9502) antibodies were obtained from Cell Signaling Technology (USA). Anti-GAPDH (sc-365062), Anti-JNK (sc-571) and Anti-IgG (sc-66931) antibodies were obtained from Santa Cruz Biotechnology (USA). Doxorubicin (D1515) and cisplatin (P4394) were obtained from Sigma-Aldrich (USA), epirubicin (56390-09-1) was obtained from Cayman Chemical (USA), Sorafenib (S7397) was obtained from Selleckchem (USA). JNK inhibitor SP600125 (S5567) was obtained from Sigma-Aldrich (USA).

WSG, a water soluble glucose-rich polysaccharide with an average molecular mass of approximately 1000 kDa, was isolated from GLPs-RF3 (Wynlife Healthcare, Inc., San Diego, CA, USA. Cat: 500T; Lot: WT20010703) as described previously [26 (link)]. Cisplatin (P4394) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). An Annexin V/PI apoptosis kit was purchased from Life Technologies (Carlsbad, CA, USA). Anti-β-actin and anti-EGFR antibodies were acquired from GeneTex (Hsinchu, Taiwan). Anti-phosphorylated AKT (S473) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-phosphorylated ERK1/2 antibody was purchased from Sigma-Aldrich (Steinheim, Germany).

Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; Ki67, p53, CyclinA2 and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig) and GAPDH (#60004-1-Ig) were from Proteintech. Fluorescence-labeled ki67 and CD133 were purchased from Abcam, USA. Recombinant human Ephrin-B2 Fc chimera protein was purchased from R&D (7397-EB, RD Inc, MN, USA). Cisplatin (P4394) was purchased from SigmaAldrich (Sigma, MO, USA). Cells were treated with cisplatin at 10 μg/ml for A549 for 48 h.

All chemicals and reagents including cisplatin (P4394) were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless noted otherwise. Media for cell culture, which includes minimum essential medium (MEM) with GlutaMAX (41090-036), Eagle’s MEM (30-2003), keratinocyte serum-free media (17005-042), and F12 medium (11765-054) were purchased from Life Technologies (Grand Island, NY, USA). The reagents for western blot were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA) and Thermo Fisher Scientific (Rockford, IL, USA). SRI110 was synthesized according to our literature report.^{38 (link)}

Cisplatin (P4394) was purchased from Sigma (USA). BCA protein assay kit (23225) was purchased from Thermo-Scientific (USA). Nitrocellulose membranes (1060003) were purchased from GE (USA). E-cadherin (#3195), Snail (#3879), Slug (#9585), Vimentin (#5741), p-ERK (#9106), ERK (#4695), and pan-keratin (#4545) antibodies were purchased from Cell signaling technology (USA). β-Actin (Sc-47778) antibody was purchased from Santa Cruz (USA). Ki-67 antibody (ab15580) was purchased from Abcam (USA). IR800 dye-conjugated rabbit (#926-32213) and mouse (#926-32212) secondary antibodies were purchased from Li-COR Biosciences (USA). Secondary antibody labeled with Cy3 (#711-165-152) was purchased from Jackson ImmunoResearch (USA) and Biotin-rabbit IgG (656140) from Invitrogen, and streptavidin-HRP (P0397) from DAKO (USA).

Cells were seeded into 96-well plates at a density of 2 × 10³ cells/well and incubated with 5% CO₂ at 37 °C for cell adherence. Each well was added with 10 uL of the CCK-8 reagent, and then, the optical density (OD) value was measured at a wavelength of 450 nm at variable time points of 24, 48, 72, and 96 h.
Then, the cell resistance was evaluated following procedures from existing research [21 (link)]. A total of 5 × 10³ cells were seeded into the 96-well plates and incubated for 24 h after transfection. Next, the cells were further incubated in variable concentrations of gefitinib (SML1657, Sigma-Aldrich Chemical Company, St Louis, MO, USA) or cisplatin (P4394, dissolved in 0.15 M NaCl, Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 48 h. The cells were incubated with the CCK-8 reagent at 37 °C for 4 h. The OD value was measured at a wavelength of 570 nm using a Microplate Reader (Sunrise Microplate Reader, TECAN, Switzerland).