Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 Page 6 of 10 lentiviral shRNAs targeting different parts of the coding sequence of BIRC6. SKNSH, one of the neuroblastoma cell lines with the highest BIRC6 expression, was trans- duced with these vectors (Figure 2a). BIRC6 is a 528 kD protein and difficult to assess with Western blot. There- fore we first analyzed BIRC6 protein levels by in cell Western. For this method cells are fixed directly in the culture well and stained with a BIRC6 antibody. Analysis shows concentration dependent down-regulation of BIRC6 protein with both BIRC6 shRNAs at 48 h after transduction (Figure 2b). Although less optimal, we can confirm BIRC6 down-regulation using Western blot Figure 2 Knockdown of BIRC6 in SKNSH induces apoptosis. a: BIRC6 mRNA expression in 24 neuroblastoma cell lines. b: In cell western of SKNSH 48 h after transduction. The Y-axis represents the ratio between BIRC6 and Actin protein expression as determined by the Odyssey bioanalyzer. The X-axis represents the concentration BIRC6 shRNA that was added. Black bars are cells transduced with control virus (SHC002), dark grey: C7 BIRC6 shRNA and light grey: C11 BIRC6 shRNA. MOI = Multiplicity of Infection. 2c: Western blot of SKNSH 48 h after transduction with no virus (NV), control virus SHC002 (C) or BIRC6 shRNA (C11 and C7). Blots were incubated with BIRC6 and actin antibodies. d: Pictures were made 72 h after transduction before protein harvest with a 100x magnitude. e: MTT-assay of SKNSH and IMR32 transduced with control virus (SHC) or BIRC6 shRNA (C7 and C11). The dark grey bars represent cells transduced with virus alone; the light grey bars represent cells that are treated with BIRC6 shRNA combined with ZVDVAD, a CASP2 inhibitor. f: Western blot of SKNSH 72 h after transduction with no virus (NV), control virus SHC002 (C) or BIRC6 shRNA (C11 and C7). Blots were incubated with PARP, DIABLO and actin antibodies.