Fig. 4

Transfection of PSMB5 Q62P mutant gene confers BTZ resistance in HEK293A cell lines. The full length PSMB5 WT and PSMB5 Q62P mutant genes were amplified from CMY and CMY-BR cell lines, cloned in pcDNA 3.1 Hygro (+) and transfected into HEK293A cells. HEK293A cells (untreated), transfected with PSMB5 WT, PSMB5 Q62P, or empty vector were treated with several concentrations of BTZ, and viability was measured and presented as percentage viable cell relative to control (untreated) (y-axis) plotted against BTZ (nM) concentrations. HEK293A cells transfected with PSMB5 Q62P showed increased resistance to BTZ compared to WT, vector only, and cells only, in a dose dependent manner (a). PSMB5 mRNA expression was amplified by qRT-PCR in CMY and BTZ-resistant CMY cell line variants (CMY BR50, CMY BR100, CMY BR200 and the CMY BR-200). The PSMB5 mRNA levels were increased significantly in the four CMY-BR variants in a dose-dependent manner. In the CMY BR-200 cells (cultured for 6 months in the absence of BTZ) PSMB5 mRNA levels decreased, but were still significantly higher than those of control (b). Expression of β5 subunit of the 20S proteasome was measured in BTZ-resistant CMY cell line variants after treatment with different concentrations of BTZ for 12 h and compared to that of the CMY parent cell line (c). The inhibition of CT-like activity was measured after 6 h of treatment with different concentrations of BTZ in CMY and the BTZ-resistant CMY cell line variants. The CT-like activity inhibition in the BTZ-resistant cells was reduced. Data are presented as percentage of the CT-like activity relative to control (y-axis) against BTZ-treatment (x-axis) (d)