*4.7. Proteasome Activity*

H2452 cells were seeded on a 96-well white plate (5 × <sup>10</sup><sup>3</sup> cells/100 <sup>μ</sup>L/well), cultured for 24 h, and subsequently treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, T3E 20 μM for 6 h, and another 6 h except for bortezomib. After the treatment, to assess chymotrypsin-like activity in cells, 50 μL of Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay Reagent (Promega Japan, Tokyo, Japan) was added to each well, and the plate was then incubated at room temperature for 20 min. The chymotrypsin-like activity was assessed based on estimated chemiluminescence intensity using a luminometer (Infinite M1000 PRO, TECAN Japan).

#### *4.8. Isolation of mRNA and Real-Time Quantitative PCR*

H2452, PANC1, and A549 cells were cultured at a density of 5 × <sup>10</sup><sup>5</sup> cells in a 60-mm dish for 24 h and were then treated with each agent for 12 to 24 h. After the treatment, cells were collected, and total RNA was isolated using the Tissue Total RNA Extraction Mini Kit (Favorgen Biotech Corp., Ping-Tung, Taiwan). Total RNA (300 ng for each sample) was used for cDNA synthesis with the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). cDNA templates were analyzed by real-time PCR using Thermal Cycler Dice Real Time System Lite (TAKARA BIO INC., Shiga, Japan) and THUNDER-BIRD™ SYBR qPCR Mix (Toyobo, Osaka, Japan), with the following program: at 95◦C for 10 s followed by 40 cycles at 95 ◦C for 15 s and at 60 ◦C for 1 min. Primer sets are shown in Table 1. Gene expression data were normalized to the expression of the reference gene ribosomal protein L32.


**Table 1.** List of PCR primers.

#### *4.9. Immunoblotting*

H2452, PANC1, and A549 cells were cultured at a density of 5 × <sup>10</sup><sup>5</sup> cells in a 60-mm dish for 24 h and then treated with each agent for 12 to 24 h. After the treatment, cells were harvested and lysed in ice-cold Laemmli sample buffer (Bio-Rad, Berkeley, CA, USA) containing a protease inhibitor cocktail (Nakadai Tesque) and phosphatase inhibitor (Nacalai Tesque). Cells were incubated on ice for 20 min following centrifugation at 12,000 rpm at 4 ◦C for 10 min. Samples were electrophoresed through a 10% or 15% SDS– polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were blocked with Blocking One P (Nacalai Tesque) for 1 h, incubated with primary antibodies for 1 h, and then incubated with the secondary antibody for 1 h. Detection was accomplished using Chemi-Lumi One Super (Nacalai Tesque) and C-DiGit (LI-COR, Lincoln, NE, USA). A densitometric analysis of each immune band was performed using Image Studio for C-DiGit (LI-COR). Molecular sizing was conducted using Protein Ladder One Plus, Triplecolor for SDS-PAGE (Nacalai Tesque). Protein concentrations were assessed using the DC Protein Assay System (Bio-Rad).
