Results: 4

1.
Figure 3

Figure 3. Wnt-3a increases total CTLA-4 protein levels in UACC 1273 melanoma cells. From: CTLA-4 Is a Direct Target of Wnt/?-Catenin Signaling and Is Expressed in Human Melanoma Tumors.

UACC 1273 cells treated with Wnt-3a-conditioned media or vehicle for 72 hours were stained with isotype control or CTLA-4 antibodies and analyzed by flow cytometry. (a) Histogram of total cellular CTLA-4 expression in fixed, permeabilized cells. (b) Histogram of surface CTLA-4 expression of live, nonpermeabilized cells. Plot is depicted as % cells of maximum versus fluorescence intensity. Dotted black, isotype control antibody for both treatment conditions; blue, vehicle, CTLA-4 antibody; red, Wnt-3a-conditioned media, CTLA-4 antibody. Data represent one of four identical experiments.

Kavita V. Shah, et al. J Invest Dermatol. ;128(12):2870-2879.
2.
Figure 2

Figure 2. CTLA-4 transcript is induced by Wnt/β-catenin signaling in two melanoma cell lines. From: CTLA-4 Is a Direct Target of Wnt/?-Catenin Signaling and Is Expressed in Human Melanoma Tumors.

(a) RT-PCR analysis of CTLA-4 transcript splice variants in A2058 melanoma cells transiently transfected with empty vector (EV) or full-length CTLA-4 (CTLA-4) and UACC 1273 and A2058 cells treated with recombinant Wnt-3a for 6 hours. (b) Real-time quantitative PCR analysis of CTLA-4 expression in UACC 1273 and A2058 cells treated with recombinant Wnt-3a or vehicle (BSA) for 6 hours. (c) Real-time quantitative PCR analysis of CTLA-4 expression in UACC 1273 and A2058 cells treated for 6 hours with Wnt-3a-conditioned media (Wnt-3a) or vehicle (control) alone or in the presence of DKK-1 conditioned media or vehicle (green fluorescent protein). Expression of CTLA-4 was normalized by comparison to GAPDH and 18S (not shown) as internal controls. Fold change is calculated relative to (b) BSA or (c) control. Gray, UACC 1273 cells; striped, A2058 cells. Data represent one of three identical experiments.

Kavita V. Shah, et al. J Invest Dermatol. ;128(12):2870-2879.
3.
Figure 1

Figure 1. CTLA-4 is expressed in human epidermal melanocytes and primary melanoma tumors. From: CTLA-4 Is a Direct Target of Wnt/?-Catenin Signaling and Is Expressed in Human Melanoma Tumors.

Dissociated human epidermal melanocytes (HEM) and two primary melanoma tumors (Mel 1, Mel 2), were analyzed for CTLA-4 (PE) and S100 (Alexa Fluor 633) expression by flow cytometry. (a) HEM, (b) Mel 1, and (c) Mel 2 were costained with the isotype control antibodies for both S100 and CTLA-4. Gates for S100- and CTLA-4-negative cell populations were set based on these results. (d) HEM, (e) Mel 1, and (f) Mel 2 were costained with S100 antibody and the isotype control antibody for CTLA-4. (g) HEM, (h) Mel 1, and (i) Mel 2 were costained with S100 antibody and CTLA-4 antibody. Blue, strongly S100-positive cells only. Red, CTLA-4-positive and strongly S100-positive cells. (jl) Histograms of CTLA-4 expression in S100 positive cells comparing CTLA4-isotype control antibody (black) with anti-CTLA-4 antibody (red) in (j) HEM, (k) Mel 1, and (l) Mel 2. Quadrant values represent percent of gated cells stained.

Kavita V. Shah, et al. J Invest Dermatol. ;128(12):2870-2879.
4.
Figure 4

Figure 4. CTLA-4 promoter is directly regulated by Wnt/β-catenin pathway through a single TCF/LEF transcription factor consensus site. From: CTLA-4 Is a Direct Target of Wnt/?-Catenin Signaling and Is Expressed in Human Melanoma Tumors.

(a) Schematic representation of CTLA-4 promoter. TCE/LEF consensus sites are numbered 1–4 with location from transcriptional start site and nucleotide sequence listed above and below, respectively. Mutated sequence is italicized. (b) A2058 cells transiently transfected with a luciferase reporter construct (WT) containing 1.7 kb (upstream of the transcriptional start site) of the CTLA-4 promoter were treated with two doses of Wnt-3a conditioned media or vehicle (control) alone or in the presence of DKK-1-conditioned media or vehicle (green fluorescent protein). (c) A2058 cells transfected with the WT reporter and mutant reporters, each containing a single mutated TCF/LEF consensus site (Mut 1–4), were treated with Wnt-3a-conditioned media or vehicle (control). CTLA-4 promoter reporter activity was normalized by cotransfection with a renilla luciferase reporter. Changes in normalized luciferase activity are represented as fold change relative to untreated reporter. Data represent three independent experiments. Error bars indicate standard deviation. Asterisk denotes statistically significant change (P<0.05) by unpaired two-tailed Student’s t-test.

Kavita V. Shah, et al. J Invest Dermatol. ;128(12):2870-2879.

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