Kar combined with Erw-ASNase results in massive mitochondria impairment. (A) LP1 and U266 cells were treated with Erw-ASNase (0.5 U/mL), Kar (3 nM), or both for 48 hours. Then cells were harvested, and whole-cell lysates were subjected to immunoblot analysis using anti–c-Myc, anti-4EBP1, anti-IRF4, or anti-GAPDH (loading control). (B) Isogenic U266 cells (pLV^empty) or c-Myc–overexpressing (pLV^cMyc-OE) cells were treated with Erw-ASNase (0.5 U/mL), Kar (0-2 nM), or both for 48 hours. Cell viability was measured using an MTS assay and is presented as a percentage of control. Inset shows immunoblot for c-Myc protein levels in tested cell lines. (C) Western blot analysis of LP1 cells shows that 24-hour treatment with Erw-ASNase (0.5 U/mL), Kar (3 nM), or both results in ER stress pathway boosting. (D) LP1 cell line was treated with Erw-ASNase (0.5 U/mL), Kar (2 nM), or combined therapy for 48 hours. Then cells were harvested, and intracellular ATP levels were measured using a sensitive enzyme cyclic assay. All data are mean ± SD of 3 independent experiments. (E) TMRE peak M2 detects signal from polarized mitochondria upon 48 hours of drug exposure (used as single agents or in combination) in U266 cells. All data are mean ± SD of 3 independent experiments. (F) Cytochrome c release from mitochondria was assessed using western blot analysis of subcellular fractions of the indicated MM cells treated with Erw-ASNase (0.5 U/mL), Kar (3 nM), or their combination for 24 hours. Then cytoplasmic and mitochondria extracts were subjected to western blotting using the indicated antibodies. The quality check for each subcellular fraction was performed using specific cytosolic (PFKP) or mitochondrial (UQRC1) markers. **P < .01, ***P < .0001, unpaired Student t test.