Pazopanib

The cell culture media RPMI, supplements and all other chemicals were purchased from Sigma Aldrich SRL (Milan, Italy). Quantitative real-time PCR reagents were acquired from Applied Biosystems (Foster City, CA, USA). Topotecan and pazopanib were bought from Selleckchem (DBA Italia, Milan, Italy), and prepared in a stock solution of 10 mM in 100% dimethylsulfoxide (DMSO) for cell's experiments. Control's medium wells were treated with the same DMSO concentration used to formulate the medium of the wells with the highest concentration of pazopanib and Topotecan in the same experiment.

Afatinib, dasatinib, pazopanib, amuvatinib, crizotinib, sunitinib, sorafenib tosylate and copanlisib were purchased from Selleckchem (Houston, TX). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Cells were purchased from the ATCC and were not further validated beyond that claimed by ATCC. Cells were re-purchased every ∼6 months. Commercially available validated short hairpin RNA molecules to knock down RNA / protein levels were from Qiagen (Valencia, CA) or were supplied by collaborators. Reagents and performance of experimental procedures were described in [15 (link)–18 (link)].

The antibodies used for western blot analysis are as follows: GAPDH (#60004-1-Ig, Proteintech, 1:5000 dilution), OTUD1 (#bs-17563R, Bioss antibodies, 1:2000 dilution), PTEN (#22034-1-AP, Proteintech, 1:1000 dilution), AKT (#bsm-33278M, Bioss antibodies, 1:2000 dilution), pAKT-S473 (#4060S, Cell signaling technology,1:1000 dilution), cleaved Caspase 3 (#9661, Cell signaling technology,1:2000 dilution), Caspase 3 (#19677-1-AP, Proteintech, 1:1000 dilution). MG132 (#S2619), Sunitinib (#S7781), Pazopanib (#S3021), JSH-23 (#S7351), MK2206 (#S1078), GSK1120212 (#S2673), and LY294002 (#S1105) were purchased from Selleckchem (Shanghai, China). HA-OTUD1 was constructed by cloning the cDNA of OTUD1 into the OmicLinkTM Expression Clone (CMV Promoter) (GeneCopoeia, EX-V0006-M14, USA). The siRNAs were purchased from RiboBio. The sequences of the siRNAs were provided in Table S1. Cells were transfected with the indicated plasmids or siRNAs using Lipofectamine 2000 (Thermo Fisher Scientific, China) according to the manufacturer's instructions. Caspase 3 activity assay kit (#ab39401) was purchases from Abcam.

Antibody for total RB was purchased from BD Biosciences (San Jose, CA, clone C245, catalog #554136). Antibodies purchased from Cell Signaling Technology (Beverly, MA) included: total RB (clone 4H1, Catalog #9309), phospho-RB (Ser608) (#2181), phospho-AKT (Ser473) (#4060), cyclin D1 (#9309), PARP-1 (#9542), caspase 3 (#9665), caspase 9 (#9502), and phospho-p70 S6K (Thr389, #9205). β-actin antibody was purchased from Sigma Aldrich (St. Louis, MO, catalog #A1978).
Laboratory-grade palbociclib was generously provided by Pfizer, Inc. (New York, NY) and purchased from Selleck Chemicals (Boston, MA). Everolimus and pemetrexed were purchased from LC chemical (Woburn, MA). Decitabine was purchased from Sigma (St. Louis, MO). Selumetinib, flavopiridol, sunitinib, AZD5438, ponatinib, AZD4547, erlotinib, pazopanib, imatinib, PF-04691502, dacomitinib and rapamycin were purchased from Selleck Chemicals (Boston, MA). Depsipeptide was obtained from the NCI Repository in the Developmental Therapeutics Program (NSC #309132, Bethesda, MD). Stock solutions for all the agents were prepared in 100% DMSO (Sigma, St. Louis, MO) at 10mM concentrations and stored at -20°C. Stock solutions were diluted in fresh RPMI 1640 media prior to each experiment.

Axitinib, cyclopamine, Erlotinib, Fasudil, Genistein, Irinotecan, Lestaurtinib, Temsirolimus, SP600125, Vatalanib, Tozasertib, Sunitinib, Dasatinib, Imatinib, Sorafenib, Nilotinib, Masitinib, PD325901 and GDC0941 were purchased from LC labs (Woburn, MA). Triptolide, 5-iodotubercidin, Calphostin C, Splitomycin, SB203580, Juglone, Thalidomide, Indirubin-3’monoxime, Compound C, Jak Inhibitor 1, Lavendustin A and FH535 were purchased from EMD (Gibbstown, NJ). Pazopanib and Regorafenib were purchased from Selleck chemicals (Houston, TX). H-89 and Rps-Be-cAMPS were purchased from Santa Cruz Biotech (Santa cruz, CA). Digitoxin was purchased from Sigma-Aldrich (St.Louis MO).