Pd166866

The drugs listed below were acquired from Selleck chem: FGFR inhibitors PD166866 (specific to FGFR1), Alofanib (targeting FGFR2), H3B-6527 (inhibiting FGFR4), and AZD4547 (effective against FGFR1-3), pan-FGFR inhibitor TAS-120, JAK inhibitors Solcitinib (inhibiting JAK1) and AZD1480 (inhibiting JAK2), UC2288 (p21 inhibitor), STAT inhibitors Fludarabine (inhibiting STAT1),
Stattic (inhibiting STAT3) and BAY2353 (Niclosamide, inhibiting STAT3), SGC-CBP30 (potent CREBBP/EP300 inhibitor), and ulixertinib (ERK1/ERK2 inhibitor). Human EGF protein and FGF ligands including FGF1, FGF2, FGF4, FGF7, FGF9, FGF8a, FG19 and FGF21 were purchased from PeproTech. The breast cancer cell lines CAMA1, MDA-MB-134, MCF7, and T47D were obtained from the American Type Culture Collection (ATCC). The CAMA1 and MCF7 cell lines were grown in DMEM with a 10% FBS and 1% antibiotic-antimycotic solution, while T47D and MDA-MB-134 were cultured in RPMI with 10% FBS and 1% antibiotic-antimycotic solution. Regular testing for mycoplasma contamination was conducted using the commercially available Myco Alert kit from Lonza. All cell lines utilized in this research have undergone authentication by ATCC, and only cells with a low number of passages were employed in experiments to ensure work confidence.

Lysates were collected in radioimmunoprecipitation assay buffer [RIPA; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritionX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 μg/mL aprotinin]. 25 μg or 30 μg of total protein was separated by 10% or 12.5% SDS-PAGE followed by transfer to Immobilon-P membrane. Immunoblotting reagents were from the following sources: antibodies against p-FGFR (Tyr653/654), FGFR1 (D8E4), FGFR4 (D3B12), p-VEGFR (19A10), VEGFR2 (55B11), p-AKT (D9E), AKT (9272), p-MAPK (D13.14.4E), p44/42 MAPK (Erk1/2), OCT4 (2750), CD133 (D2V8Q), Androgen Receptor (D6F11), and β-actin (4967) antibodies were from Cell Signaling Technology; FGFR2 (C-8), FGFR3 (B-9), and STAT5 (C-17) were from Santa Cruz Biotechnology; ALDH7A1 (CAT:ABO11656) was from Abgent; HRP anti-mouse, HRP anti-rabbit, and Enhanced Chemiluminescence (ECL) reagents were from GE Healthcare. Other reagents included: Dovitinib, BGJ398 and PD166866 were from Selleckchem.

H9C2 cells (Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in high glucose DMEM (GIBCO, USA) supplemented with 1% cyan streptomycin double-antibody and 10% (v/v) FB Sat 37°C under a 5% CO₂ atmosphere. For the present study, H9C2 cells were incubated overnight to reach 70-80% confluence at 37°C before experimentation. H9C2 cells were treated with H₂O₂ at a concentration of 100 μM for 4 hours to construct an apoptosis model [26 (link)]. AMPK was activated after exercise [27 (link)], so AICAR, an AMPK agonist, was used to simulate the exercise effect of H9C2 cells. Furthermore, H9C2 cells were treated with recombinant human FGF21 (rhFGF21, 75 ng/ml, 15 h; Selleck, USA), FGFR1 inhibitor (PD166866, 100 ng/ml, 15 h, Selleck Chemicals) [8 (link)], PI3K inhibitor (LY294002, 10 μM, 1 h, Selleck Chemicals), AICAR (1 mM, 1 h, Selleck Chemicals) [28 (link)], and lentiviral vectors carrying alcat1 gene (ALCAT1 OE, MOI = 1; Brain VTA, China).

Mouse FGF2 is a recombinant protein purchased from Prospec (CYT-386). Fresh powder for confirmation was ordered from the NCI’s DTP or from Caymen Chemical. Picropodophyllin and fluvastatin were obtained from the DTP (Fig. 2). Fluvastatin (10010337), simvastatin (MK-733), rosuvastatin (ZD 4522), pravastatin (10010342), atorvastatin (10493), cycloheximide (14126), and mitomycin C (11435) were ordered from Caymen Chemical. PD166866 (S8493) and NVP-ADW742 (S1088) was purchased from SelleckChem. eBioscience™ Cell Proliferation Dye eFluor™ 450 (65-0842-85) and SYTOX™ Red Dead Cell Stain, for 633 or 635 nm (S34859) were purchased from ThermoFisher. I-BET-762 was purchased from JSTAR Research Inc.