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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 5. — TFAP2A and TFAP2C functionally support the ΔNp63-mediated activity of the MIR944 promoter. (A) ChIP-qPCR results for binding of ΔNp63 to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. (B) Keratinocytes were transfected with a MIR944 promoter-luciferase construct or a p63-binding site #3 mutant of the MIR944 promoter-luciferase construct. After 5 days of incubation in high-calcium medium, differentiated keratinocytes were lysed, and 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 Ca treatment 0 day, unpaired Student's t-test. (C) ChIP-qPCR results for binding of TFAP2A (left panel) and TFAP2C (right panel) to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (D) The effect of TFAP2A or TFAP2C overexpression on the ΔNp63-mediated activities of the MIR944 promoter was examined. WM266-4 melanoma cells were transfected with a MIR944 promoter-luciferase construct along with the indicated expression vectors. 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, unpaired Student's t-test. (E) Reduction in the expression of miR-944 with TFAP2A or TFAP2C depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. TFAP2A and TFAP2C mRNA levels were normalized to RPLP0 expression, and miR-944 expression was normalized to that 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. (F) Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. The following day, keratinocytes were transfected with a 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.

Figure 5. — TFAP2A and TFAP2C functionally support the ΔNp63-mediated activity of the MIR944 promoter. (A) ChIP-qPCR results for binding of ΔNp63 to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. (B) Keratinocytes were transfected with a MIR944 promoter-luciferase construct or a p63-binding site #3 mutant of the MIR944 promoter-luciferase construct. After 5 days of incubation in high-calcium medium, differentiated keratinocytes were lysed, and 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 Ca treatment 0 day, unpaired Student's t-test. (C) ChIP-qPCR results for binding of TFAP2A (left panel) and TFAP2C (right panel) to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (D) The effect of TFAP2A or TFAP2C overexpression on the ΔNp63-mediated activities of the MIR944 promoter was examined. WM266-4 melanoma cells were transfected with a MIR944 promoter-luciferase construct along with the indicated expression vectors. 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, unpaired Student's t-test. (E) Reduction in the expression of miR-944 with TFAP2A or TFAP2C depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. TFAP2A and TFAP2C mRNA levels were normalized to RPLP0 expression, and miR-944 expression was normalized to that 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. (F) Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. The following day, keratinocytes were transfected with a 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.