Extended Data Fig. 4: Detection of telethonin expression by flow cytometry in patient-derived cells treated with SpCas9.
From: Precise therapeutic gene correction by a simple nuclease-induced double-stranded break
a, Contour plots from a representative flow cytometry assay to detect telethonin expression in healthy control cells (TCAP+/+), patient cells (TCAP−/−), and SpCas9-treated homozygous and heterozygous iPS clone-derived myoblasts differentiated for 10 days in culture. Plots are representative of three independent replicates. b, Histograms from a representative flow cytometry assay to detect telethonin expression. Left, overlay of anti-telethonin antibody staining for four representative samples for different TCAP genotypes. Right, comparison between patient cells and healthy control cells, and SpCas9-treated homozygous and heterozygous iPS clone-derived myoblasts differentiated for 10 days in culture. Histograms are representative of three independent replicates. c, Cells were selected by removing cell debris first as shown by gate P1, and then single cells were selected from P1 by removing clustered cells as shown by gate P2. The cells in gate P2 were used for flow analysis. Plots are representative of one biological replicate. d, Average percentage of telethonin-expressing cells from two technical replicates of three biological replicates. Error bars indicate s.e.m (n = 6) and circles represent individual data points. P values (P = 0.33 for patient versus heterozygous and *P = 0.04 for patient versus homozygous clones) were calculated by two-sided Student’s t-test (Supplementary Table 9). ns, not significant. e, Western blot showing validation of anti-telethonin antibody (Santa Cruz Biotechnology). Human muscle lysate and lysate from HEK293T cells transfected with haemagglutinin-tagged telethonin expression construct were separated on an SDS 4–12% acrylamide gradient gel and the resulting blot was probed with anti-telethonin antibody. For gel source data, see Supplementary Fig. 1.
