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. 2022 Apr 28;13:2306. doi: 10.1038/s41467-022-30015-1

Fig. 4. Expression of frameshift variants causes increased cell toxicity during C2C12 differentiation.

Fig. 4

a Schematic of C2C12 differentiation. Differentiation media consists of DMEM supplemented with 0.5% FBS, 1% penicillin/streptomycin, and 1% l-glutamate. b Representative images of C2C12 cells one (top) or two (bottom) days after change to differentiation media from three independent experiments. Scale bar, 20 μm. c Representative scatter plots of GFP-positive C2C12 cells stained with propidium iodide (PI) and annexin V, one (top) or two (bottom) days after change to differentiation media. d Quantification of GFP-positive cells that were negative for both PI and annexin V. Values represent means ± s.e.m. (n = 3 independent experiments). ***P = 0.0004, **P = 0.0061, and **P = 0.0052 (day 1) and *P = 0.0116, *P = 0.0252, and *P = 0.0147 (day 2) for N323Tfs*36, G328Afs*31, and G331Efs*28 mutants, respectively, by two-way ANOVA with Dunnett’s multiple comparisons test. e Quantification of cell survival for all GFP-positive cells from day 1 post differentiation split into three categories. The range of GFP intensity was 500–140,000. Cells whose GFP intensity was below 45,000, between 45,000 and 90,000, or above 90,000 were grouped into low, medium, or high GFP-expressing cells, respectively. Values represent means ± s.e.m. from n = 3 independent experiments. *P = 0.0364 and **P = 0.0088 by one-way ANOVA with Tukey’s multiple comparisons test.