Figure 7. Effect of LPS and Ubc9 overexpression on Ubc9, Gclc and Gclm expression in mouse hepatocytes.

Primary cultures of mouse hepatocytes were treated with Ubc9 overexpression vector or empty vector (V) and then subjected to LPS treatment as described in Methods. Part A) shows the effect of Ubc9 overexpression on basal Gclc and Gclm mRNA levels and after LPS treatment for 4 hours. Ubc9 overexpression raised Gclc (but not Gclm) mRNA level and prevented LPS-mediated lowering of both. Results are expressed as % of V from three experiments, *p<0.05 vs. V, †p<0.05 vs. LPS. Part B) shows the same treatments as A) on protein levels of the same genes after 18 hours. Representative Western blots are shown and densitometric changes are expressed as % of V in the box below. Results are from three experiments, *p<0.02 vs. V, †p<0.05 vs. LPS. Part C) shows the effect of LPS on Ubc9 protein level is not due to increased protein degradation. Hepatocytes were treated with cycloheximide prior to LPS for 4 to 18 hours. Graph on the right summarizes changes in densitometry of Ubc9 as a function of time, expressed as % of 4-hour control. Results are from 3 experiments, *p<0.05 vs. 4 hour control, †p<0.05 vs. 18-hour control.