Bortezomib reduces HLA class I expression on JJN3 and primary MM in a dose- and time-dependent fashion. MM cells were incubated with various doses of bortezomib for indicated times. PI-negative cells were gated and analyzed for class I expression. (A) Dose response: JJN3 was exposed to increasing doses of bortezomib for 24 hours. (B) Time course: JJN3 cells were incubated at 10 nM bortezomib for increasing durations of time. (C) Dose response: Patient MM was exposed to increasing doses of bortezomib for 24 hours. (D) Time course: Patient MM was incubated at 10 nM bortezomib for increasing durations of time. Class I expression was also tested on JJN3 after bortezomib by spinning-disc confocal microscopy. (E) Dose response: JJN3 was exposed to increasing doses of bortezomib for 24 hours. (F) Time course: JJN3 cells were incubated at 10 nM bortezomib for increasing durations of time. Images were acquired using a Zeiss Axiovert 200M microscope (Carl Zeiss) fitted with a BD Bioimaging CARV II spinning-disc confocal accessory. Mid-cell confocal images were presented (original magnification, ×250). The fluorescence intensity was quantified using IPLab version 3.9.5 software and corrected by substracting the blank field intensity. A total of 30 cells per group from 3 independent experiments were considered and the mean intensity (± SD) was plotted. (G) The kinetics of apoptosis on JJN3 after drug treatment. JJN3 was treated with 10 nM bortezomib for indicated times. Cells were stained with annexin V and PI and analyzed by FACS. The data represent 1 of 3 individual experiments. The percentages are shown of cells that are annexin V and PI double negative (lower left quadrant), double positive (upper right quadrant), solely annexin V positive (lower right quadrant), and solely PI positive (upper left quadrant).