Ctip2 directly regulates Notch1 expression in differentiated cells.

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Ctip2 directly regulates Notch1 expression in differentiated cells. Ctip2 directly regulates Notch1 expression in differentiated cells. (A) Subconfluent keratinocytes were cultured in growth medium (GM) or in starvation medium (SM) without added growth factors for 24 hrs before RT-qPCR analyses for K10 mRNA expression. Results shown are means ± s.e.m. (n = 3; normalized to GAPDH; ***P<0.0001). (B) RT-qPCR analyses of Notch1 mRNA levels (normalized to GAPDH) in keratinocytes cultured in GM or SM conditions. Results are shown as means ± s.e.m. (n = 3; *P<0.01). (C) Keratinocytes cultured in GM or SM conditions were subjected to immunoblotting analyses using anti-Notch1 and β-actin antibodies. (D) Starved keratinocytes were treated with or without 10 ng/ml EGF for 24 hrs before being subjected to RT-qPCR analyses of K10 expression (normalized to GAPDH). Results are shown as means ± s.e.m. (n = 3). (E) Starved keratinocytes treated with or without EGF were subjected to RT-qPCR analyses of Notch1 mRNA expression (normalized to GAPDH). Results are shown as means ± s.e.m. (n = 3). (F) Immunostaining of the E14.5 and E16.5 skin sections with an anti-Notch1 antibody (red); nuclei were counterstained with DAPI (blue). Scale bar: 10 µm. (G) Notch1 protein expression was analyzed by immunoblotting of whole cell extracts from E14.5 and E16.5 skin using anti-Notch1 and anti-β actin antibodies. (H) Starved keratinocytes treated with 0.2 mM CaCl2 (HiCa) for the indicated times were subjected to ChIP analyses using anti-Ctip2 antibody or IgG (rat) as a control. The purified chromatin was analyzed by qPCR using primers covering the distal promoter, the proximal promoter or 3′ UTR region of the Notch1 gene. Each bar represents the ChIP signals using anti-Ctip2 relative to signals from non-specific IgG. (I) Starved keratinocytes were treated with 10 ng/ml EGF for the indicated times, and then subjected to ChIP analyses as described above. Statistical significance was determined using Student's unpaired t-test. The results are representative of at least three independent studies. Ling-juan Zhang et al. J Cell Sci 2012;125:5733-5744 © 2012. Published by The Company of Biologists Ltd