Glucocorticoid receptor knockdown and GSH content in B16 melanoma cell subsets; and plasma corticosterone, ACTH, and IL-6 levels during melanoma growth <i>in vivo</i>.
<p>(A) GCR levels were measured by Western blot in control metastatic iB16 melanoma cells isolated from the liver and their equivalents stably expressing GCR-shRNA. Similar blots were run for B16-F10 and B16-F10-shGCR growing <i>in vitro</i>. Each lane in the blots corresponds to an individual representative animal in the indicated group. The relative density of each band was normalized against the internal standard (β-actin) on each blot (n = 4–5 in all cases) and expressed as relative changes in arbitrary densitometry units. Results obtained in cells transfected with lentiviral vector not harboring any gene (negative control) were not different from control values (not shown). *p<0.01 <i>versus</i> iB16 cells. <i>In vivo</i> experiments show data obtained after 7 days of inoculation. <i>In vitro</i> experiments show results obtained in cells cultured for 72h. (B–D)Blood was collected from the tail vein during a 24-h period starting 7 days after tumor inoculation, and peak plasma levels of corticosterone and ACTH (6 h and 12 h, circadian time, respectively) measured. Melanoma cells were isolated before GSH determination. Tumor volume and GSH levels were measured 8 days after inoculation. Data are mean values ± S.D. of 7–8 different animals. *p<0.05, **p<0.01 <i>versus</i> controls.</p
