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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Nat Med. 2014 Nov 24;20(12):1452–1457. doi: 10.1038/nm.3736

Figure 3. T835M UNC5C increases cell death beyond that induced by WT UNC5C.

Figure 3

Representative growth curves of (a) GFP+ cells (transfected only) and (b) total cells (transfected and non-transfected) (c) Growth of βgal-, T835M-, or WT-expressing cells. GFP+ specific growth was normalized to total growth, and slopes of the best-fit lines were calculated. Data represents four independent transient transfection experiments with n=6 randomly plated technical replicates per condition for each experiment. ***p=0.007, ** p=0.0011, *p=0.0259. (d) Representative flow cytometry plots of βgal-, WT- and T835M UNC5C-transfected cells analyzed for GFP and AnnexinV-APC; untransfected cell population (orange), GFP+ AnnexinV- cell population (green), and GFP+ AnnexinV+ population (black). (e) Percentage of AnnexinV+ cells within the GFP+ population representing four independent transient transfection experiments with 100,000 cells analyzed per condition. ***p< 0.0001, ** p=0.0014, *p=0.0111. (f) Representative overlay of fluorescent and bright field images from time-lapse imaging experiments; transfected cells (red), caspase-3/7 activity (green); Scale bar = 50 μM (g) Average lifetime of cells expressing βgal (n=919), WT UNC5C (n=687), or T835M UNC5C (n=590) at movie start or that initiated expression during first 20 h of imaging. (h) Kaplan-Meier survival curves of cells expressing βgal (grey, n=1696), WT (blue, n=1085), or T835M UNC5C (red, n=751) analyzed by Wald test for p-values. (g–h) Data represent three independent transient transfection experiments with n=6 randomly plated technical replicates per experiment and 24 movies per genotype, ***p< 0.0001. Data was analyzed with experimenter blinded to samples, represents the mean ± SEM, and was analyzed by two-tailed unpaired t-test.