Fig 3. BIRC6 mediates drug resistance in MYL-R cells independently of Mcl-1.

(A) Anti-BIRC6 shRNAs were used to knock down BIRC6 in MYL-R cells. Immunoblot analyses showed that shBIRC6-58, -59, and -61 yielded efficient knockdown without affecting Mcl-1. (B) MYL-R cells were resistant to imatinib (IC50 ~3.0 μM) as compared to MYL cells (IC50 ~0.2 μM), and (C) BIRC6 knockdown sensitized MYL-R cells to imatinib (IC50 ~0.2 μM). MYL, MYL-R, and BIRC6 knockdown MYL-R cells were cultured in triplicate in 96-well plates with increasing concentrations of imatinib for 72 hours, and cell viability was assessed by MTS assay. (D) Treatment of BIRC6 knockdown MYL-R cells with imatinib showed elevation in caspase-3/7 activity. BIRC6 knockdown MYL-R cells were treated with 1 μM imatinib for 24 hours. Parental MYL-R cells were treated with DMSO or 1 nM dasatinib or 1 μM imatinib. Caspase-3/7 activity was measured using a fluorogenic substrate as described in Materials and Methods. (E) BIRC6 knockdown or imatinib treatment in MYL-R cells did not affect total Lyn or total Mcl-1 protein levels. BIRC6 knockdown MYL-R cells were treated with DMSO or 300nM or 1 μM imatinib (IM) for 24 hours, and immunoblot analyses used to measure total Lyn and total Mcl-1 proteins. (F) Knockdown of BIRC6 in MYL-R cells increased sensitivity to gemcitabine. BIRC6 knockdown MYL-R cells were cultured as described in (C) with increasing concentrations of gemcitabine for 72 hours and cell viability determined by MTS assay. The data presented in this figure are representative of at least three independent experiments, and * represents p < 0.05.