FIGURE 3.

Effects of 1,25(OH)₂D₃ with or without UO126 treatment on nuclear localization of endogenous VDR and RXRα in HPK1A and HPK1ARas cells. HPK1A cells were grown and treated with either vehicle (Veh) or 1,25(OH)₂D₃ (1,25D3). The cells were also fixed and stained with VDR-Alexa-488 (A) or RXRα-Alexa-488 (C) antibodies to detect VDR or RXRα. Alexa-488 (C) followed by treatment with either vehicle (veh) or 1,25(OH)₂D₃. Hoechst dye was used as a DNA marker as discussed under “Experimental Procedures.” Similarly, HPK1ARas cells were grown and treated with either vehicle or 1,25(OH)₂D_3. The cells were then fixed and stained with VDR-Alexa-488 (E) or RXRα-Alexa-488 (G) antibodies to detect VDR or RXRα. Hoechst dye was also used as a DNA marker as discussed under “Experimental Procedures.” Nuclear localization was assessed as under “Experimental Procedures.” Scatter plots show the quantitation of fluorescence of nuclear receptors normalized to total cell fluorescence (B, D, F, and H). Values represent mean ± S.D. of at least 10 different cells. Asterisks indicate a significant difference in nuclear localization between 1,25(OH)₂D₃ treatment alone compared with vehicle-treated control. Open circles indicate a significant difference in nuclear localization of receptors in 1,25(OH)₂D₃-treated cells alone compared with combined UO126 and 1,25(OH)₂D₃ treatment. A p value of p < 0.05 was considered significant.