Figure 2.

Celastrol inhibits NOS activity in CRC cells. (A) HCT116 cells were treated with celastrol (2, 3 μM) or vehicle control for 24 h. The NOS activity was tested by measuring nitrate concentration. No significant change of medium nitrate concentration after celastrol administration was observed. (B) HCT116 cells were treated with celastrol (2, 3 μM) or vehicle control for 24 h. Cell lysates were extracted with cell lysis buffer and were subjected to NOS assays. (C) HCT116 cells were treated with iNOS shRNA plasmids for 48 h, and the iNOS knockdown cell population was selected with puromycin treatment for 3 days. The cells with stable knockdown of iNOS were then treated with 2 μM celastrol or vehicle control for 24 h and cell proliferation analyzed by MTS assay. (D) The HCT116 cells with stable knockdown of eNOS were treated by adding 2 μM celastrol or vehicle control for 24 h and then the cell proliferation was analyzed by MTS assay. All data are shown as the mean ± SEM. *p < .05, **p < .01, ***p < .001 vs. control group; #p < .05 vs. eNOS knockdown group. CEL = celastrol. iNOS KD = shRNA-knockdown iNOS. eNOS KD = shRNA-knockdown eNOS.