Supplementary Figure 4: BRN2 regulation of a diverse repertoire of targets to control neuronal maturation.
From: Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells
(a) Characterization of hNPCs by immunostaining Nestin (Green), BRN2 (Red) and SOX2 (Red).
(b) Immunofluorescence analysis of neurons derived from hNPCs after BRN2 knockdown.
(c) The impact of BRN2 knockdown on the expression of MAP2 (Green) and SOX2 (Red) in neurons derived from hNPCs. n= 3 independent experiments for (a), (b) and (c). Scale bar: 20 µm.
(d) Immunostaining of NeuN after knockdown of BRN2 in PTB/nPTB sequentially depleted HAFs. n=3 random 20x fields.
(e) Impaired Na+ (Red) and K+ (Blue) currents in BRN2 knockdown neurons from hNPCs.
(f) Western blotting analysis of MAP2, Tuj1 and BRN2 in differentiated hNPCs. GAPDH served as loading control. n= 2 biological repeats. Uncropped versions of Western blots are shown in Supplementary Figure 7.
(g) Test of 4 commercially available anti-BRN2 antibodies by immunoprecipitation. C-20 and B-2 were chosen for ChIP-seq library construction. GAPDH served as input control. Uncropped versions of Western blots are shown in Supplementary Figure 7.
(h, i) GO term analysis of BRN2 target genes. Both GO terms of biological processes (g) and biological components (h) indicate BRN2 is critical for neuronal maturation. Background was based on all expressed genes in hNPCs.
