Extended Data Fig. 6: Pro-neuronal activity of Myod1 upon co-expression of multi-lineage repressor Myt1l.

a, Representative immunofluorescence images of reprogrammed cells with MAP2 (red), MYH (green) and DAPI (blue) 14 days after TF-induction. Scale bar: 100 µm. b, Single cell quantification of MAP2+ cells with neuronal morphology (left) and MYH+ cells (right) in a. n = 3 biologically independent samples. c, Normalized expression levels of neuronal and muscle marker genes 14 days post TF induction, determined by qRT-PCR. n = 3 biologically independent samples. d, Western blot of reprogramming MEFs in a, two days post TF induction. e, Representative immunofluorescence images of MEFs reprogrammed with either Ascl1+Myt1l+Gfp or Myod1+Myt1l+Gfp for six weeks on primary mouse glia. Scale bar: 50μm. f, Quantification of the fraction of TUJ1+/GFP+ neurons found in e that are vGAT or VGLUT positive. Violin plots indicate the median and interquartile range. n = 4 technical replicates. (g,j) Representative immunofluorescence images of reprogrammed MEFs 14 days post Ascl1 (g) or Myod1 (j) induction together with either Gfp, Myt1l full length, or a non-functional Myt1l (410-623) truncation that contains two DNA binding zinc fingers fused with either an engrailed repressor (EnR) domain or a VP64-activator (VP64) domain. Scale bar: 100 µm. (h,k) Single cell quantification of TUJ1+ cells with neuronal morphology (green) and DES+ cells (red) in g,j upon addition of Myt1l full length or Myt1l-EnR fusion constructs to either Ascl1 (h) or Myod1 (k) alone. n = 3 biologically independent samples. (i,l) Western blot of reprogramming MEFs in g,j two days post-induction of Ascl1 (i) or Myod1 (l) in combination with indicated constructs. All immunofluorescence and western blots were repeated 3 times with similar results, except ED Fig. 6e, which was only repeated twice; Unprocessed blots ED Fig. 6; For all bar graphs, error bars = SEM, two-tailed Student’s t-test, * p < 0.05, ** p < 0.01; *** p < 0.001.

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