PI3K pathway is active at baseline in 8-chloroadenosine resistant, but not in sensitive, cell lines.

<p><b>a</b>. RPPA heatmap showing proteins that are differentially expressed between the most sensitive and resistant cell lines at baseline prior to treatment. AKT phospho S473 and S308 were higher and phospho AMPK T172, ACC S79, and total PTEN were lower in the resistant cell lines. <b>b</b>. Western blots showing phospho and total AKT and S6 levels in sensitive and resistant cells before and after 40 μM 8-chloroadenosine treatment. Beta-actin served as loading control. <b>c</b>. Western blots showing phospho ERK T202/204 levels in sensitive and resistant cells before and after 40 μM 8-chloroadenosine treatment. Beta-actin served as loading control. <b>d</b>. Quantification of phospho AKT, S6, and ERK and total AKT and S6 levels.</p

mTOR pathway downregulation in sensitive cell lines upon treatment with 8-chloroadenosine.

<p><b>a</b>. RPPA heatmap showing expression changes that correlate with IC₅₀ values. ccRCC cells were treated with 40 μM 8-chloroadenosine for 24 hours. Graphs show changes in phospho S6 levels in sensitive and resistant cell lines. p-values are calculated using two-tailed paired t-test. coef. Pearson correlation coefficient between the IC₅₀ values and levels of proteins measured by RPPA. <b>b</b>. Western blot validating phospho S6 S235/236 downregulation in CAKI1 and ACHN cell lines upon 8-chloroadenosine treatment for 24 hours. <b>c</b>. Western blot showing phospho ACC S79 as readout of AMPK activity and phospho p70S6K T389 and phospho S6 S235/236 for mTOR pathway activity. ccRCC cells were treated with 40 μM 8-chloroadenosine for 1, 5, 10, 18, 35, 60, 120, or 300 minutes. Vinculin served as loading control.</p

Decrease in ATP and AMPK activation in sensitive and resistant cell lines.

<p><b>a.</b> ATP levels in sensitive and resistant ccRCC cell lines after 6 hours of 40 μM 8-chloroadenosine treatment. ATP levels decreased significantly in all cell lines (CAKI1 p<0.01, ACHN p = 0.02, RCC4 p<0.01, RXF393 p<0.01); however, ATP levels were not significantly different between the sensitive and resistant cell lines (p = n.s.). <b>b.</b> Western blots for phospho AMPK T172 and AMPK activity readout phospho ACC S79 after 0, 8, 16, and 24 hours of treatment with 40 μM 8-chloroadenosine. Vinculin is used as loading control.</p

Efficacy of 8-chloroadenosine in ccRCC cell lines.

<p><b>a.</b> ccRCC cell lines treated with 20 μM or 80 μM 8-chloroadenosine for 7 days. <b>b.</b> Cell cycle analysis of ccRCC cell lines treated with 40 μM 8-chloroadenosine for 48 hours. <b>c.</b> IC₅₀ values of ccRCC cell lines calculated in Calcusyn software.</p