Fig. 8.

Downregulation of lentiviral-expressed CYP2C22 by NO in HuH7 cells is not attenuated by inhibitors of calpain or lysosomal degradation. (A) Effect of calpain inhibitors on CYP2C22 downregulation. Fully confluent 3-day-old HuH7 cells stably expressing CYP2C22 in 12-well plates were treated with DPTA (500 μM) and/or E64d (10 μM), MDL-28170 (MDL, 10 μM), or ALLM (10 μM) for 4 hours, and total cell lysates were harvested for CYP2C22 and GAPDH immunoblotting. a, Significantly different from control, untreated cells; b, significantly different from cells treated with inhibitor alone, P < 0.05, ANOVA and Tukey’s test (n = 3). (B) Inhibition of calpain activity. Cells cultured as described in (A) were treated with control media or calpain inhibitors E64d (10 μM), MDL-28170 (MDL, 10 μM), and ALLM (10 μM) for 2 hours. The media were exchanged for assay buffer containing the calpain substrate Boc-LM-CMAC (25 μM), and fluorescence in the media was measured after 20 minutes of incubation. *, Significantly different from control; P < 0.05, ANOVA and Dunnett’s test (n = 4). (C) Effect of lysosomotropic agents. To study lysosome-dependent degradation of annexin VI, cells were first deprived of serum for 4 hours to enhance the expression of annexin VI, and then serum was added to stimulate its degradation. DPTA (500 μM) and/or chloroquine (CQ, 100 μM) or NH₄Cl (20 mM) were added concomitantly with the serum, and the cells were incubated for an additional 4 hours. Total cell lysates were harvested for Western blotting. a, Significantly different from control, untreated cells; b, significantly different from cells treated with inhibitor alone, P < 0.05, ANOVA and Tukey’s test (n = 3).