Fig. 1

DARPP-32 promotes cell survival and negatively regulates apoptosis. a NSCLC A549, b H1650 and c H226 cell lines were transduced with lentivirus encoding LacZ control shRNA or DARPP-32 shRNAs (clone numbers 03 and 04). DARPP-32 and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates. Immunoblots are representative of three independent experiments. d A549, e H1650 and f H226 cells transduced with control or DARPP-32 shRNAs were seeded into 60 mm culture dishes for 16 h. Flow cytometry-based apoptosis assays were performed following incubation with anti-annexin V antibodies conjugated with APC. g A549, h H1650 and i H226 cells were transduced with control or DARPP-32 shRNAs and immunoblotted with antibodies to detect cleaved and uncleaved PARP, cleaved and uncleaved (i.e., pro-) caspase-3, DARPP-32 and α-tubulin (loading control). j A549 and k H226 cells were transduced with retrovirus containing control (LacZ), DARPP-32- or t-DARPP-overexpressing clones. DARPP-32, t-DARPP and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates. Immunoblots are representative of three independent experiments. l A549 and m H226 cells transduced with control, DARPP-32- or t-DARPP-overexpressing clones were seeded into 96-well cell culture plates for 72 h. Colorimeter-based cell survival assay was conducted using MTS reagents. Each open circle on a graph represents an independent experiment. Uncropped images of depicted immunoblots are shown in Supplementary Figs. 13-15. Error bars indicate SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison