Skip to main content
. 2020 May 11;11(5):353. doi: 10.1038/s41419-020-2566-1

Fig. 4. SC66 induces oxidative stress, SphK1 inhibition, and JNK activation in RCC cells.

Fig. 4

786-O cells were treated with MK-2206, LY294002 or plus SC66 (all at 3 μM), cells were further cultured, and cell viability was tested by MTT assay (a, 72 h). Stable 786-O cells with AKT1 shRNA (“sh-AKT1”) or CRISPR/Cas9 AKT1-KO construct (“ko-AKT1”), as well as the control cells with scramble control shRNA and Cas9 empty plasmid (“scr-shRNA+Cas9-c”), were tested by Western blotting assay of AKT expression (b, the upper panel), cells were treated with/without SC66 (3 μM) for 72 h, cell viability was tested (b, the lower panel). 786-O cells or the primary human RCC cells (“RCC1”) were treated with SC66 (3 μM) for indicated time periods, ROS production (c and k), GSH/GSSG ratio (d), mitochondrial depolarization (e and l), SphK1 expression and activity (f), as well as the ceramide contents (g and m) and p-/t-JNK expression (h and n) were tested by the appropriate assays. 786-O cells were pretreated for 30 min with NAC (400 μM), JNKi (10 μM), or S1P (10 μM), followed by SC66 (3 μM) treatment for 48 and 72 h, cell viability and apoptosis were tested by MTT assay (h) and TUNEL staining assay (i), respectively. Phosphorylated JNK1/2 was normalized to total JNK1/2 (h and n). For each assay, n = 5. Data were expressed as the mean ± standard deviation (S.D.). *P < 0.05 vs. “Veh” group. #P < 0.05 vs. SC66 treatment only (i, j). In this figure, experiments were repeated three times, and similar results were obtained each time.