Figure 2. Characterization of RLS-7 derivatives.

(a) Structures of RLS-7 derivatives. (b) A375 melanoma cells were treated with RLS-7 and indicated derivatives for 6 h under hypoxia. Whole cell lysates were immunoblotted with indicated antibodies. Quantification of immunoblots was performed using BioRad densitometer, relative to loading controls, noted under the blots (c) A375 cells were treated with different concentrations of indicated compounds and cell viability was assessed by ATPlite after 72 h. Each bar represents the mean ± standard deviation from three experiments. Representative data is shown. ****p < 0.0001 compared to the control (one-way ANOVA with Dunnett’s test). (d) Different melanoma cells were treated with RLS-12 and cell viability was assessed by ATPlite after 72 h. ****p < 0.0001 based on comparison with control using one-way ANOVA, as described above. (e) Prostate cancer cells RV1 and PC3 were treated with different concentrations of RLS-7 or RLS-12, and cell viability was assessed by ATPlite after 72 h. ****p < 0.0001 based on comparison with control using (one-way ANOVA with Dunnett’s test). (f) A375 cells were plated at low density and grown in medium containing vehicle, 2 µM or 10 µM of RLS-7, or RLS-12. The num ber of colonies formed after 10 days in culture was determined by crystal violet staining. (g) Immunohistochemistry for the expression of AR and chromogranin A (CHGA) of PCa tumors from castrated mice treated with RLS-12. Scale bar indicates 100 µm. (h) A375 cells treated with vehicle, 2 µM or 10 µM of RLS-12 for 24 h under hypoxia. mRNA was extra cted and subjected to qPCR analysis of indicated HIF target genes. Means ± standard errors were calculated based on two independent experiments. ***p < 0.001, ****p < 0.0001 were calculated based on comparison with DMSO-treated control using Student’s t test.