*2.3. T3, TOS, and T3E Suppressed the Recovery of Proteasome Activity under and after Bortezomib Treatment*

Under or following proteasome inhibition, NFE2L1 is known to recover proteasome activity [14]. We hypothesized that T3, TOS, and T3E might inhibit the recovery of proteasome activity under or after proteasome inhibition. Subsequently, we next evaluated the effects of TP, T3, TOS, and T3E on the recovery of proteasomal chymotrypsin-like activity undergoing and following bortezomib treatment. Proteasome activity was significantly reduced in all treatment groups compared to the control group (Figure 3a). After removing bortezomib from each treatment group, proteasome activity recovered to approximately 60% in the bortezomib alone group, whereas it was less than 40% in the combination groups with bortezomib and T3, TOS, T3E (Figure 3b).

Since ubiquitinated proteins are well known to accumulate under proteasome inhibition, T3, TOS, and T3E may enhance the accumulation of ubiquitinated proteins induced by PIs via inhibition of the recovery in proteasome activity. For this reason, we next evaluated the effect of the combination of bortezomib with TP, T3, TOS, and T3E on the accumulation of ubiquitinated protein by an immunoblot. As seen in Figure 3c, the combination groups with bortezomib and T3, TOS, and T3E showed a more remarkable accumulation of ubiquitinated protein compared to the bortezomib alone group. These results suggest that T3, TOS, and T3E inhibit the recovery of proteasome activity under and after proteasome inhibition.
