Fig. 2
From: SETD5 haploinsufficiency affects mitochondrial compartment in neural cells
Mitochondrial damage in Setd5+/− NSCs. A Control and mutant NSCs (two different clones, C5 and D3) immuno-stained with TOMM-20 mitochondrial marker. Nuclei are stained with Hoechst 33,342. dashed rectangles identify the magnification shown on the right (scale bar = 10 micron). The level of circularity, as calculated by ImageJ macro (see methods) between 0–1 is shown. Statistics, one-way ANOVA (P = 0.0001), multiple comparison Dunnet test (Ctrl vs. Clone C5, P = 0.0023; Ctrl vs. Clone D3, P < 0.0001) B Top, ultrastructural analysis by transmission electronic microscopy (TEM) of mitochondria in Setd5+/− (Mutant) and control (WT) NSCs (scale bar = 500 nm). Quantification of the mitochondria major axis length is shown on the right. Statistics, Mann–Whitney test (P < 0.0001). Bottom, quantification of the different kind of mitochondria retrieved as indicated. Statistics, one-way ANOVA (P = 0.0001), multiple comparison Tuckey test (Ctrl vs. Mutant, P < 0.0001; Ctrl vs. Mutant, P < 0.0001; Ctrl. vs. Mutant, P = 0.0325; Ctrl vs. Mutant, P = 0.0257). C Western blot analysis on whole protein lysate from control and Setd5+/− NSCs (two different clones, C5 and D3) for the indicated mitochondrial protein, normalized on total Tomm-20 level. Relative quantification on the right. Statistics; NDUFS4 (Complex I) one-way ANOVA (P = 0.0002), multiple comparison Dunnet test (Ctrl vs. Clone C5, P = 0.0006; Ctrl vs. Clone D3, P < 0.0002); NDUFB8 (Complex I), one-way ANOVA (P = 0.0002), multiple comparison Dunnet test (Ctrl vs. Clone C5, P = 0.0006; Ctrl vs. Clone D3, P < 0.0002); SDHB (Complex II), one-way ANOVA (P = 0.0672), multiple comparison Dunnet test (Ctrl vs. Clone C5, P = 0.1025; Ctrl vs. Clone D3, P = 0.0647); UQCRIII (Complex III), one-way ANOVA (P < 0.0001), multiple comparison Dunnet test (Ctrl vs. Clone C5, P < 0.0001; Ctrl vs. Clone D3, P < 0.0001); MTCO1 (Complex IV), one-way ANOVA (P = 0.0265) multiple comparison Dunnet test (Ctrl vs. Clone C5, P < 0.0338; Ctrl vs. Clone D3, P < 0.0278); ATP5A (Complex V) ANOVA (P = 0.0003) multiple comparison Dunnet test (Ctrl vs. Clone C5, P = 0.0002; Ctrl vs. Clone D3, P = 0.0032). D, E Both control and mutant NSCs (three different clones, B4, C5, and D3) were treated with cell-permeant fluorescent dyes that are either insensitive (Mitotracker green, D) or sensitive (Mitotracker orange, E) to mitochondrial membrane potential. Flow cytometry analysis estimate either mitochondrial mass using mitotracker green (D) or mitochondrial potential with the orange (E). Mitotracker Green. Statistics, one-way ANOVA (P = 0.3081), multiple comparison Dunnet (Ctrl vs. Clone C5, P = 0.3142; Ctrl vs. Clone D3, P < 0.3051). Mitotracker Orange. Statistics, one-way ANOVA (P = 0.0068), multiple comparison Dunnet (Ctrl vs. Clone C5, P = 0.0100; Ctrl vs. Clone D3, P < 0.0067. All Data are presented as mean values+/− SEM