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1.
Fig. 2.

Fig. 2. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

ΔNp63α does not inhibit TAp63-mediated induction of VDR. (A) H1299 cells were transfected with either empty vector or TAp63γ alone or along with increasing concentrations of ΔNp63α as indicated. 24 hours after transfection, TaqMan-based real-time PCR was performed to detect the transcript levels of VDR, p21 and IGFBP-3. (B) H1299 cells were transfected with either empty vector or TAp63γ and/or ΔNp63α as indicated. Immunoblot analysis was performed using anti-VDR and anti-p63 isoform and anti-β-actin (used as a loading control) specific antibodies.

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.
2.
Fig. 7.

Fig. 7. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

p63 is essential for VDR expression during embryonic development. (A) Total RNA from E18.5 wild-type MEFs and p63–/– MEFs were extracted and levels of p63, VDR and Id2 mRNA were determined by using TaqMan-based real-time PCR analysis. Detection of Id2 expression was used as nonspecific target for p63. (B) Immunofluorescence expression analysis of VDR (green) and p63 (red) from paraffin-embedded skin sections from p63-null mice and newborn (NB), postnatal day 4 (P4) and 16 (P16) wild-type mice.

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.
3.
Fig. 4.

Fig. 4. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

Silencing of ΔNp63α results in reduced VDR expression. (A) A431 cells were transfected with either control siRNA (siCon) or three different siRNAs against p63. 48 hours after transfection, TaqMan-based real-time PCR was performed to detect the transcript levels of VDR and p63. The y-axis represents the fold change in VDR and p63 mRNA transcript levels relative to control-siRNA-transfected cells. (B) A431 cells were transfected with either control siRNA or three different siRNAs against p63 as indicated. 48 hours after transfection, whole-cell lysates were subjected to immunoblot analysis using anti-p63, anti-VDR and anti-β-actin (used as loading control) specific antibodies.

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.
4.
Fig. 6.

Fig. 6. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

Overexpression of ΔNp63α inhibits cell invasion. (A) A431-eGFP, A431-ΔNp63α and A431-VDR stable pool cells were subjected to Matrigel-based invasion assay (top panel). The y-axis represents the fold change in number of stable pool cells invaded, compared with A431-eGFP stable pool cells. Immunoblot analysis was performed to detect the expression of p63 and VDR in A431-eGFP, A431-ΔNp63α and A431-VDR stable pool cells (bottom panel). (B) A431-eGFP, A431-ΔNp63α and A431-VDR stable pool cells were transfected with either control siRNA or p63 siRNA as indicated. 24 hours after siRNA transfection, cells were subjected to Matrigel-based invasion assay. The y-axis represent the fold change in number of cells invaded compared with control A431-eGFP cells, and error bars represent the s.d. from two independent experiments. The bottom panel shows staining of the lower membrane of the invasion chamber from a representative experiment under these conditions.

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.
5.
Fig. 1.

Fig. 1. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

Transcriptional activation of VDR by all p63 isoforms. H1299 (A) and HeLa (B) cells were transfected with either empty vector, or expression plasmids encoding all the isoforms of p63 as indicated. 24 hours after transfection, total RNA was extracted and subjected to TaqMan based real-time PCR to detect the transcript levels of VDR and p21. The y-axis represents fold change in VDR and p21 transcript levels relative to empty vector transfected cells (left panels). 24 hours after transfection, cells were harvested for whole-cell extracts and subjected to immunoblot analysis using anti-VDR, anti-p21, anti-p63 and anti-β-actin (used as a loading control) antibodies (right panels).

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.
6.
Fig. 5.

Fig. 5. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

Downregulation of ΔNp63α or VDR leads to increased cell migration and invasion. A431 cells were transfected with either control siRNA (Con si), p63 siRNA or VDR siRNA as indicated. (A) After 24 hours, cells were initiated for wound healing assay and microscopic pictures were taken at 0 hours and 24 hours after initiation of wound-healing assay (left panel). Quantification of the distance between the edges of the wound normalized to 100% at 0 hours is represented as wound closure as shown on the right panel. Values represent mean of three independent experiments. (B) Cells were subjected to Matrigel-based invasion assay. At 24 hours after plating, images of cells migrated through the Transwell membranes were taken and total number cells migrated through membranes was counted. The y-axis represents the fold change in number of cells invaded through membrane in cells transfected with p63 siRNA or VDR siRNA compared with control siRNA, and error bars represent the s.d. from three independent experiments.

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.
7.
Fig. 3.

Fig. 3. From: Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion.

VDR is a direct target of ΔNp63α. (A) H1299 cells were co-transfected with full-length VDR promoter-Luc reporter construct along with either control vector or increasing concentrations of ΔNp63α as indicated. 24 hours after transfection, cells were subjected to dual luciferase assay. The y-axis represents fold change in relative luciferase units (RLU) compared with cells transfected with empty vector. (B) Whole-cell extracts from H1299 cells transfected with the indicated p63 isoform and from A431 cells were subjected to immunoblot analysis with pan-p63 (4A4), ΔNp63-specific (RR-14) and p63α-specific (H-129) antibodies. Overexpressed ΔNp63α in H1299 corresponding to the endogenous ΔNp63α in A431 cells is denoted with an asterisk. (C) A431 cells were subjected to chromatin immunoprecipitation assay. Crosslinked chromatin was immunoprecipitated using two different antibodies against p63 and control IgG as indicated. Eluted DNA was then subjected to PCR amplification of VDR promoter, and amplification of p21 promoter was used as a positive control for p63.

Ramakrishna Kommagani, et al. J Cell Sci. 2009 Aug 15;122(16):2828-2835.

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