Figure 3.

Reduction of nuclear foci following DOX-induced gene editing in DM1-derived proliferating and differentiated cells

Cells were infected with the indicated lentiviral combinations and cultured in proliferation medium (GM) or differentiation medium (DM) for 7 days (D7) with or without doxycycline (DOX), then fixed and processed for RNA FISH analysis using a fluorescent (CAG)₆CA probe and MYOG staining (only for differentiated cells). (A) Representative images of untreated and DOX-treated EF1-Crispr-transduced cells in GM and DM, stained as indicated, are shown. White arrows highlight edited nuclei without foci. Scale bar, 10 μm. Note that treated myotubes contain both foci-negative and foci-positive nuclei. (B) Histograms show the increase of both total (left panel) and MYOG-positive (right panel) foci-free nuclei following DOX treatment. (C and D) Histograms show quantitation of (C) total nuclei containing no foci, ≤5 foci, and >5 foci in growing cells (GM) and (D) no foci, ≤5 foci, between 6 and 20 foci, and >20 foci in differentiated cells (DM) transduced with EF1-Crispr following DOX treatment. (E and F) Histograms show quantitation of MYOG-positive nuclei containing no foci, ≤5 foci, between 6 and 20 foci, and >20 foci in differentiated cells (DM) transduced with (E) EF1-Crispr or (F) CK8-Crispr following DOX treatment. At least 300 nuclei were counted for each condition (mean ± SEM). n = 3–4. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.