Xenbase Image ID: 152495

Figure S4. Tctp knockdown compromises axonal mitochondrial function. (A) Stage 32 control and Tctp morphant eye explants were grown in vitro for 24 hours and incubated with MitoTracker to visualize mitochondria. The plot shown here represents the average number of axonal mitochondria found in con-MO- or tctp-MO-positive retinal ganglion cells (boxplot: whiskers cover 5-95 percentile, ‘+’ denotes the mean value; n = no. of axons analysed; *** P < 0.0001, Mann-Whitney test). (B) Plot of axonal mitochondrial length in control and Tctp-depleted retinal ganglion cells (boxplot: whiskers cover 5-95 percentile, ‘+’ denotes the mean value; n = no. of axons analysed; n.s., P = 0.7960, Mann-Whitney test). (C-E) Normal pgc1a expression in HCT116 cells deficient for Tctp. The rational for applying this seemingly indirect approach is not immediate. We knew a priori that basal P53 levels were elevated in tctp+/- mice (Amson et al., 2012) and that the expression of pgc1a was repressed by the activation of P53 in the context of telomere dysfunction (Sahin et al., 2011). Since the mitochondrial traits identified in Tctp morphants were reminiscent of dysfunctional Pgc1α activity (Wareski et al., 2009), we hypothesised that the activation of P53 in Tctp-deficient backgrounds could result in mitochondrial dysfunction due to its repression of pgc1a expression. Hence the strategy used here, for it allowed us to evaluate the expression of pgc1a at the transcriptional level. (C) The promoter region of human pgc1a (2.8 kb) was cloned upstream of the luciferase gene cassette and stably transfected into tctp or control shRNA-infected HCT116 cells. Subsequently, lysates of tctp or control shRNA-infected HCT116 cells were run on a SDS-PAGE gel and Tctp protein expression verified by western blot. Tctp expression knockdown was as high as 85% in these cells (D). We show in (E) a plot of the average relative light units (RLU) measured in cells co-transfected with pgc1a-luc and a control plasmid (mean ± SEM; six biological replicates of tctp-shRNA-infected cells were measured in triplicate in the two independent rounds of experiments conducted; *** P = 0.5544, one-way ANOVA). (F) Quantitative analysis of mitochondrial transport velocities. Population-wide study of mitochondrial velocities (excluding stationary organelles) suggests that no significant differences exist between control and Tctp-depleted axons, in agreement with the dataset shown in Fig. 7I (mean ± s.e.m.; anterograde direction: n.s., P = 0.9941; retrograde direction: n.s., P = 0.9371; statistical analyses used Mann-Whitney test). Image published in: Roque CG et al. (2016) © 2016. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

Return to previous page