Figure 2.
KMT2B Is Essential for MEF-to-iN Transition
(A) Efficiency of transdifferentiation in ScanR experiments, calculated relating, for any chosen concentration, the number of Tuj1+ at 13 days to the number of DAPI 3 days after plating.
(B) Cell mortality in ScanR experiments, calculated as the ratio of DAPI 3 days (DAPI start) and 13 days (DAPI end) after plating.
(A and B) Kmt2a+/+Kmt2b+/+, n = 3; Kmt2bfl/fl, n = 2; Kmt2afl/flKmt2bfl/fl, n = 1.
(C) Efficiency of transdifferentiation in FACS experiments, calculated relating the number of PSA-NCAM+ cells at 13 days to the number of plated cells.
(D) Percentage of dead cells, calculated at 13 days, over the initial number of plated MEFs.
(C and D) The number of independent embryos per genotype corresponds to the one reported for time point 13 days of (E).
(E) Percentage of PSA-NCAM+ cells among transdifferentiating MEFs 5, 7, 9, 13, and 21 days after plating, assayed with FACS analysis. The number of independent embryos per time point per genotype is reported under the graph.
(F) Representative images of three different experiments, each reporting the specific cKO and its control. Kmt2afl/− (top left), Kmt2a+/+Kmt2b+/+ (top, center and right), Kmt2a−/− (bottom left), Kmt2b−/− (bottom center), and Kmt2a−/−Kmt2b−/− (bottom right) iNs at 13 days are reported (DAPI in blue and Tuj1 in red).
(G) Representative images of 21-day Kmt2a+/+Kmt2b+/+ (left), Kmt2a−/− (center), and Kmt2b−/− (right) iNs acquired with the bright-field microscope.
(H–J) Average neurite length per neuron, calculated with Neuritetracer on day 13 of transdifferentiation. In particular Kmt2a−/− iNs (H), Kmt2b−/− iNs (I), and Kmt2a−/− Kmt2b−/− iNs (J) were compared to controls.
In the case of Kmt2a ScanR experiments (F and H), Kmt2afl/− MEFs were used as a control. In (F), (H), and (J), MEFs were plated for BAM transduction 2 days after 4-OHT, whereas, in (A)–(E), (G), and (I), MEFs were plated 7 days after 4-OHT. (A)–(E) and (H)–(J), means ± SEM; ∗∗∗p < 0.0001; ∗p < 0.01; ns, not significant.