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. 2015 Jul 21;43(15):7462–7479. doi: 10.1093/nar/gkv735

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — ΔNp63 is responsible for the activation of the MIR944 promoter. (A) ChIP-seq results obtained from GEO (accession number GSE32061) for the MIR944 promoter for two independent biological replicates. (B) ChIP-qPCR results for ΔNp63 binding to the MIR944 promoter in keratinocytes that had been transfected with the si-NC compared to those transfected with TP63 siRNA (si-p63). Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (C) Reduction in miR-944 expression with p63 depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TP63 mRNA (si-p63) or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. The levels of ΔNp63 mRNA were normalized to the expression of RPLP0, and those of miR-944 were normalized to levels of RNU48 small RNA. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (D) WM266-4 melanoma cells, which do not express ΔNp63 and miR-944, were transfected with the ΔNp63 promoter-luciferase or the MIR944 promoter-luciferase construct in combination with the pcDNA (mock vector), ΔNp63α, ΔNp63β or ΔNp63γ expression vectors. After 1 day of incubation, luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus pcDNA, unpaired Student's t-test. (E) Keratinocytes were transfected with 50 nM siRNA against TP63 mRNA (si-p63) or si-NC. After 1 day, the keratinocytes were transfected with the MIR944 promoter-luciferase construct. After 2 days of incubation, keratinocytes were lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (F) UCSC Genome Browser view of the PhyloP score and the nucleotide alignment of multiple species for the predicted p63-binding sites on the MIR944 promoter region. In the bottom, the sequences of the wild type and the mutant type were shown. (G) Each of three predicted p63-binding sites was mutated by site-directed mutagenesis. WM266-4 melanoma cells were transfected with a wild type or mutant type MIR944 promoter-luciferase construct in combination with a pcDNA (mock vector), ΔNp63α, or ΔNp63β expression vector. After 1 day of incubation, the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus pcDNA, unpaired Student's t-test.

Figure 4. — ΔNp63 is responsible for the activation of the MIR944 promoter. (A) ChIP-seq results obtained from GEO (accession number GSE32061) for the MIR944 promoter for two independent biological replicates. (B) ChIP-qPCR results for ΔNp63 binding to the MIR944 promoter in keratinocytes that had been transfected with the si-NC compared to those transfected with TP63 siRNA (si-p63). Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (C) Reduction in miR-944 expression with p63 depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TP63 mRNA (si-p63) or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. The levels of ΔNp63 mRNA were normalized to the expression of RPLP0, and those of miR-944 were normalized to levels of RNU48 small RNA. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (D) WM266-4 melanoma cells, which do not express ΔNp63 and miR-944, were transfected with the ΔNp63 promoter-luciferase or the MIR944 promoter-luciferase construct in combination with the pcDNA (mock vector), ΔNp63α, ΔNp63β or ΔNp63γ expression vectors. After 1 day of incubation, luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus pcDNA, unpaired Student's t-test. (E) Keratinocytes were transfected with 50 nM siRNA against TP63 mRNA (si-p63) or si-NC. After 1 day, the keratinocytes were transfected with the MIR944 promoter-luciferase construct. After 2 days of incubation, keratinocytes were lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (F) UCSC Genome Browser view of the PhyloP score and the nucleotide alignment of multiple species for the predicted p63-binding sites on the MIR944 promoter region. In the bottom, the sequences of the wild type and the mutant type were shown. (G) Each of three predicted p63-binding sites was mutated by site-directed mutagenesis. WM266-4 melanoma cells were transfected with a wild type or mutant type MIR944 promoter-luciferase construct in combination with a pcDNA (mock vector), ΔNp63α, or ΔNp63β expression vector. After 1 day of incubation, the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus pcDNA, unpaired Student's t-test.