Fig. 5.

Mchanistic investigation of c-Myc, HIF-1α, and Sp4 in the regulation of AS expression in melanoma cells under arginine-available and -deficient conditions. A. Western blotting analyses of expression levels of AS, HIF-1α, c-Myc and Sp4 in A2058, SK-MEL-2 and A375 cells grown in the regular, ADI-containing (0.05 µg/ml), and arginine-free medium for 72 hours. B – D: Chromatin immunoprecipitation assay of the interactive transcriptional regulators with AS promoters under arginine depletion conditions. B and C, c-Myc displaces HIF-1α binding from AS promoter in response to arginine depletion in A2058 and SK-MEL-2 cells, respectively. D, Dissociation of HIF-1a from but no concomitant association of c-Myc to the AS promoter in response to arginine depletion in A375 cells. E, Stabilization of HIF-1α by the hypoxia mimic CoCl₂ resulted in persistent association between HIF-1α and the AS promoter and preventing c-Myc to interact with the promoter in A2058 cells cultured under arginine-deficient conditions The antibodies used for ChIP and control IgG are indicated at the top. Input, genomic DNA prior to immunoprecipitation. AS promoter sequences from ChIP were quantified by PCR assay.