Figure 5.

Delayed Induction of Neurog2ERT2 Reveals a Block in Astrocyte Reprogramming

(A) Scheme of the experimental procedure.

(B and C) Micrographs of Neurog2ERT2-infected astrocytes (red) immunostained for GFAP (green) and βIII-tubulin (white), without (B) or with (C) OHT treatment starting at 6 days after being plated. Scale bars, 100 μm.

(D) Histogram depicting the proportion of GFAP+ or βIII-tubulin+ cells among infected cells upon delayed Neurog2ERT2 activation at 13 DPI. n = 4 independent experiments.

(E and F) Histograms of real-time qPCR (E) and HA-Neurog2ERT2 μChIP-PCR (F) of astrocyte cultures treated as indicated in the legend (early, early OHT treatment, gray bars in E from Figure 1A; and delayed, OHT treatment 6 days later). For (F) cells were exposed to OHT treatment for 24 hr. Percentages of input chromatin were quantified in duplicate from three independent biological samples (mean ± SEM). Significance was tested between samples and respective Dll1 ORF negative region by two-tailed unpaired t test (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.0001).

(G) Scheme of the experiment.

(H–J) Micrographs of astrocytes transfected with the constructs indicated on top of the panels with a 5 day delay immune-stained for βIII-tubulin 8 days post transfection (DPT). Scale bars, 50 μm.

(K) Histogram depicting the proportion of βIII-tubulin+ cells at 8 DPT. Mean ± SEM; n = 4 independent experiments (^∗p < 0.05).

See also Figure S5.