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. 2016 Apr 28;25(13):2681–2697. doi: 10.1093/hmg/ddw127

Figure 7.

Figure 7.

Knockdown of C9orf72 rescues TMEM106B-induced vacuolar phenotype. (a, b) In HeLa cells (a) and in HEK293 cells (b), treatment with control siRNA does not affect the LAMP1+ vacuolar phenotype seen in TMEM106B over-expressing cells (arrows, top row), but siRNA knockdown of C9orf72 mitigates the phenotype (bottom row). For all panels, the right panel shows the merged channels for TMEM106B (green) and LAMP1 (red); individual channels are shown in monochrome in the left and middle panels. Scale bars = 10 µm. TMEM106B detected by N2077 antibody. (c, d) The diameter of LAMP1+ organelles was quantified in HeLa (c) and HEK293 (d) cells. In the left graph for both cell types, LAMP1+ diameter was assessed under endogenous conditions (first column), TMEM106B over-expression (second column), TMEM106B over-expression with C9orf72 knockdown (third column), and TMEM106B over-expression with control siRNA knockdown (fourth column). In the right graph, LAMP1+ diameter is quantified under endogenous conditions (first column), C9orf72 over-expression (second column), C9orf72 knockdown alone (third column), TMEM106B over-expression alone (fourth column), and TMEM106B and C9orf72 concomitant over-expression (fifth column). All values are normalized to the endogenous condition (first column) and means ± SEM from nine replicates performed on three separate days are shown. In both HeLa (c) and HEK293 (d) cells, TMEM106B over-expression significantly increased LAMP1+ organelle size (p < 0.0001, left graph, compare first and second columns). In both cell types, however, knockdown of C9orf72 abrogated the effects of TMEM106B over-expression, resulting in a return of LAMP1+ organelle size toward that of the endogenous baseline (p < 0.001, left graph, comparing third column with second and fourth columns). ****p < 0.0001, ***p < 0.001.