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. Author manuscript; available in PMC: 2022 Jan 15.
Published in final edited form as: J Cell Sci. 2021 Jul 23;134(14):jcs256388. doi: 10.1242/jcs.256388

Fig. 4. mRNA distribution patterns are maintained upon translation inhibition.

Fig. 4

(A) Representative FISH heatmaps of total mRNA in myofibers treated for 6 h with 200 μg/ml puromycin (Puro) or 20 μg/ml cycloheximide (Chx). Images on the right show 1.5× magnifications of the perinuclear region (undashed boxes) and 50 μm away from the nucleus (dashed boxes). (B) Quantification of poly(A) intensity at different distances from the nucleus in control, puromycin- and cycloheximide-treated cells (color-coded as in A), normalized to the average intensity near the nucleus in untreated cells of each experiment. Mean±s.e.m. of 40 (control), 48 (Puro) and 47 (Chx) cell segments from three independent experiments. Statistical significance presented at 0 μm, 25 μm and 50 μm relative to control. (C) PEI of total mRNA in control myofibers (Ctrl) and myofibers treated for6 h with puromycin or cycloheximide. Mean±s.e.m. of 40, 48 and 47 cell segments, respectively, from three independent experiments. (D) Representative smFISH images of the distribution of Actn2, Cacna2d1 and Ubb mRNA in myofibers treated for 6 h with puromycin or cycloheximide. Images on the right show 1.5× magnifications of the perinuclear region (undashed boxes) and 50 μm away from the nucleus (dashed boxes). (E) Quantification of Actn2, Cacna2d1 and Ubb mRNA amount relative to distance to the nucleus in control, puromycin- and cycloheximide-treated cells (color-coded as in F–H). Mean±s.e.m. of 28, 19 and 26 (Actn2); 21, 21 and 23 (Cacna2d1); and 29, 30 and 28 (Ubb) cell segments, respectively, from three independent experiments. Statistical significance presented at 0 μm, 25 μm and 50 μm relative to control. (F-H) PEI of Actn2 (F), Cacna2d1 (G) and Ubb (H) mRNA in control, puromycin and cycloheximide-treated cells. Mean±s.e.m. of 28, 19 and 26 (Actn2); 21, 21 and 23 (Cacna2d1); and 29, 30 and 28 (Ubb) cell segments, respectively, from three independent experiments. (I) Representative smFISH images of the distribution of Ttn, Ryr1 and Macf1 mRNA in myofibers treated for 6 h with puromycin and cycloheximide. Images on the right show 1.5× magnifications of the perinuclear region (undashed boxes) and 50 μm away from the nucleus (dashed boxes). (J) Quantification of Ttn, Ryr1 and Macf1 mRNA amount relative to distance to the nucleus in control and puromycin and cycloheximide-treated cells (color-coded as in K–M). Mean±s.e.m. of 22, 24 and 20 (Ttn); 27, 21 and 24 (Ryr1); and 22, 18 and 20 (Macf1) cell segments, respectively, from three independent experiments. Statistical significance presented at 0 μm, 25 μm and 50 μm relative to control. (K–M) PEI of Ttn (K), Ryr1 (L) and Macf1 (M) mRNA in control and puromycin- and cycloheximide-treated cells. Mean ±s.e.m. of 22, 24 and 20 (Ttn); 27, 21 and 24 (Ryr1); and 22, 18 and 20 (Macf1) cell segments, respectively, from three independent experiments. ***P<0.001; **P<0.01; *P<0.05; ns, P>0.05 (one-way ANOVA with Tukey’s multiple comparisons test). smFISH images are maximum intensity projections. Scale bars: 10 μm.