Abt 199

GBM cells (1 × 10⁴) were plated in 96 well plates and treated with inhibitors of Calmodulin (CGS-9343B, Sigma), Casein kinase II subunit alpha (Emodin, Sigma), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (U73122, Sigma), Tyrosine-protein kinase ABL1 (Dasatinib, Santacruz) and B-cell lymphoma 2 (ABT199, Santacruz) for 24hr and subsequently incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT,100 μg/ml DMSO) in fresh culture medium for 3 hr. Optical density (OD) was taken at 550 nm with an ELISA reader (Thermo) as described elsewhere [105 (link)].

(R)-4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl)amino)-3 ((trifluoromethyl) sulfonyl) phenyl) sulfonyl) benzamide (ABT-263); 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[3-nitro-4-(oxan-4 ylmethylamino) phenyl] sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy) benzamide (ABT-199) (Santa Cruz Biotechnology, Santa Cruz, CA); 5-[3-[4-(aminomethyl)phenoxy]propyl]-2-[(8E)-8-(1,3-benzothiazol-2-ylhydrazinylidene)-6,7-dihydro-5H-naphthalen-2-yl]-1,3-thiazole-4-carboxylic acid (WEHI-539) (a generous gift from Genentech USA Inc, San Francisco, CA, USA) and PFT-μ (Sigma, St. Louis, MO, USA) were dissolved in DMSO and stored at -20°C. Inhibitors were added to cell cultures such that the final DMSO concentrations were kept constant at 0.25% (v/v).

Lapatinib ditosylate and ABT-199 were purchased from Santa Cruz Biotechnology (Heidelberg, Germany); cobalt (II) chloride hexahydrate from Sigma Aldrich (Schnelldorf, Germany); trastuzumab (Herceptin®) from Roche (San Francisco, CA, USA); and ZVAD pan-caspase inhibitor z-VAD-fmk from Bachem (Bubendorf, Switzerland). Antibodies against human Mcl-1 (S-19), extracellular signal-regulated kinase 2 (ERK2), and Ubiquitin (P4D1) were obtained from Santa Cruz Biotechnology; antibodies against Her2/ErbB2 (D8F12), Poly (ADP-ribose) polymerase (PARP), Bcl-xL, and Prohibitin-1 from Cell Signaling Technology (Boston, MA, USA); the antibody against α-tubulin from Sigma Aldrich; the antibody against Hif-1α, EpCAM, and ErbB2/Her2 from BD Biosciences (Heidelberg, Germany); the antibody against Bcl-2 (ab18210) from Abcam (Boston, MA, USA); and the antibody against murine Mcl-1 from Rockland Immunochemicals Inc., Limerick, PA 19468, USA.