Figure 6. FOXD3 Regulates Transcription of VISTA mRNA.
(A) A375 cells were treated with doxycycline for 96 h to induce the overexpression of a DNA-binding mutant FOXD3. VISTA and FOXD3 levels were detected by western blot. Data are representative of 3 independent experiments.
(B) A375 cells were treated with doxycycline for various lengths of time to induce the expression of either WT or DNA-binding mutant FOXD3.
(C) Cells were treated with doxycycline for varying lengths of time to induce FOXD3 expression. RNA was isolated and qPCR was performed using 2 different Taqman assay probes for VISTA mRNA. Data are representative of 3 independent experiments. *p < 0.05, **p < 0.01.
(D) ChIP-seq data of V5-FOXD3 were mined for FOXD3 binding peaks. Shown is a map of the locus for the VISTA gene VSIR and aligned reads. Data were visualized in the Integrated Genomics Viewer.
(E) FOXD3 binding at identified peaks was verified by ChIP. A375TR/V5-FOXD3 cells were treated with doxycycline for 48 h to induce V5-FOXD3 expression. V5 antibody was used to pull down FOXD3 protein, and specific primers (Table S2) were used to probe binding at each peak (labeled in 5D) by qRT-PCR. Data are representative of 3 independent experiments. *p < 0.05, **p < 0.01.