The FL118 downstream target survivin play a role in FL118-mediated inhibition of cancer cell growth and apoptosis.

<p><b>a</b> Subconfluent HCT-8 colon cancer cells were infected with a lentiviral delivery system containing mock shRNA (shRNA-EGFP) or survivin shRNA. After puromycin selection at 2 µg/ml, the puromycin-selected infectants were treated with or without FL118 for 72 hours. Cells were then analyzed using the MTT assay for cell viability. Each bar is the mean ± SD derived from three independent assays. Of note, downregulation of survivin by the lentiviral survivin shRNA was confirmed by western blots (Fig. 5d). <b>b–d.</b> Subconfluent HCT-8 colon cancer cells were infected with lentiviral survivin shRNA particles or control lentiviral particles as above. After up to 7 days selection with puromycin (2 µg/ml), the mixed infectants were treated with or without FL118 (10 nM) for 36 hours. Cells were stained with Annexin V/PI, followed by flow cytometry. <b>b.</b> A representative flow cytometry result gated with PI (Y axis) and Annexin V (Alexa Fluor 647, X axis). <b>c</b> and <b>d.</b> Quantitative data from <b>b</b> for R4 (c) and R2+R4 (d) from three independent measures in parallel. Of note, R1 is Annexin V negative/PI positive cells; R2 is both Annexin V and PI positive cells (later apoptotic cells); R3 is both Annexin V and PI negative cells; and R4 is Annexin V positive/PI negative cells (early apoptotic cells).