**2. Results**

### *2.1. T3, TOS, and T3E Suppressed the Increase in Protein Levels of NFE2L1 Induced by Bortezomib Treatment*

NFE2L1 is known to stabilize and translocate from cytoplasm to nucleus under proteasome inhibition [30]. First, we confirmed the protein levels of NFE2L1 in the cytoplasm and nucleus by immunoblotting. For solid cancer, we used the malignant pleural mesothelioma, H2452, due to its reported resistance to bortezomib [13,31]. As shown in Figure 1a, two bands were identified in the cytoplasmic fraction under bortezomib treatment, and the lower band was clearly identified in the nuclear fraction. Therefore, we assumed that the upper band is unprocessed NFE2L1 without transcriptional activity and the lower band is processed NFE2L1 with transcriptional activity.

Next, we used an immunoblot to evaluate the effect of TP (α-tocopherol), T3, TOS, and T3E on the protein levels of NFE2L1 under bortezomib treatment. As shown in Figure 1b, bortezomib alone demonstrated a significant increase in protein levels of unprocessed and processed NFE2L1 compared to the control group, while the combination group with bortezomib and T3, TOS, and T3E did not show this increase. Also, TOS, and T3E alone groups showed a trend toward a decrease in NFE2L1 protein level. These results suggest that T3, TOS, and T3E modulate protein levels of NFE2L1 under proteasome inhibition.

#### *2.2. T3, TOS, and T3E Suppressed the Increase in Expression Levels of Proteasome-Related Proteins Induced by Bortezomib Treatment*

It is known that the expression levels of proteasome-related proteins are increased by NFE2L1 under proteasome inhibition [14,32]. We also speculated that T3, TOS, and T3E might also moderate expression levels of proteasome-related proteins under proteasome inhibition. Consequently, we next evaluated the effects of TP, T3, TOS, and T3E on the expression levels of the proteasome-component proteins, PSMA7, PSMB7, and PSMC4, as well as POMP, a proteasome maturing protein, under bortezomib treatment by RT-PCR assay. As demonstrated in Figure 2a, the bortezomib alone group showed a significant increase in the mRNA expression levels of each protein compared to the control group, while the combination groups of bortezomib with T3, TOS, and T3E did not show as significant an increase as that in the bortezomib alone group. These results suggest that T3, TOS, and T3E modulate the expression levels of proteasome-related proteins under proteasome inhibition.

**Figure 1.** Effects of vitamin E on protein level of NFE2L1 in treatment of bortezomib. (**a**) H2452 cells were treated with bortezomib (50 nM) for 12 h and fractionated into cytoplasm and nucleus by NE-PER™ nuclear and cytoplasmic extract reagent kit. Protein levels in both fractions were assessed by immunoblotting. (**b**) H2452 cells were treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, and T3E 20 μM for 12 h, and the protein levels were assessed of NFE2L1 by immunoblotting. α-Tubulin protein levels served as the loading control. A densitometric analysis was performed as described in the Materials and Methods section. Data are means ± SD, n = 3. \* *p* < 0.05, \*\* *p* < 0.01 vs. as indicated. BTZ; bortezomib, TP; α-tocopherol, T3; α-tocotrienol, TOS; α-tocopheryl succinate, T3E; 6-O-Carboxypropyl-alpha-tocotrienol.

**Figure 2.** Effects of vitamin E on mRNA level of proteasome related proteins in the treatment of bortezomib. H2452 cells were treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, T3E 20 μM for 12 h, and the mRNA levels of PSMA7, PSMB7, PSMC4, and POMP were assessed by real-time quantitative PCR as described in the Materials and Methods section. RPL32 mRNA levels served as the loading control. Data are means ± SD, n = 3. \*\* *p* < 0.01 vs. as indicated. BTZ; bortezomib, TP; α-tocopherol, T3; α-tocotrienol, TOS; α-tocopheryl succinate, T3E; 6-O-Carboxypropyl-alpha-tocotrienol.
