YK-135 inhibits mitochondrial OXPHOS complex I activity. (A) Assessment of OXPHOS functions for YK-135. Basal, maximal, ATP-linked respiration and proton leak in 2 EMT (SNU484 and HGC27) and 2 non-EMT (MKN45 and NCI-N87) gastric cancer cell lines 24 hours post-YK-135 treatment by a Seahorse bioanalyzer. P-values were determined by Student’s t test (**P < 0.01, ***P < 0.0001, n.s.: not significant). (B) SNU484 cells were treated with DMSO or YK-135 (10 μM) for 24 h. Cells were permeabilized using plasma membrane permeabilizer (PMP), and OCR was measured with sequential administration of a combination of pyruvate (10 mM), malate (0.5 mM), and ADP (4 mM) for complex I-linked respiration or rotenone (1 μM), succinate (10 mM) and ADP (4 mM) for complex II-linked respiration. (C) Complex I enzymatic activity was measured from mitochondrial fractions of SNU484 cells. Mitochondrial fractions were isolated and then treated with YK-135 or rotenone in vitro. (D) Selective sensitivity of OXPHOS inhibitors in 7 EMT (HGC27, SK4, SNU668, MKN1, YCC11, SNU484, and Hs746T) and 5 non-EMT cell lines (NCI-N87, MKN45, SNU719, SNU216, and NUGC4). Boxplots represent mean of relative viability 72 h post-treatment of YK-135 (5 μM), phenformin (0.63 mM) or rotenone (0.16 μM). P-values were determined by Student’s t test.