Fig. 3

Relationship between PKM2 and KITENIN. A–B CaCo2/EV and CaCo2/KITENIN cells were transfected with si-PKM2 and then their metabolism was analyzed. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using a Glycolytic Rate Assay Kit on a Seahorse XF96 extracellular flux analyzer. To assess the glycolytic rate, the assay utilizes both ECAR and OCR measurements to determine the glycolytic proton efflux rate (glycoPER). GlycoPER was measured at two time points, and then following the sequential injection of rotenone (1 μM)/antimycin A (1 μM) and 2-DG (50 mM). C–D Invasion assay for CaCo/EV and CaCo2/KITENIN cells transfected with si-negative control or si-PKM2, using fibronectin as a chemoattractant. KITENIN-overexpressing cells showed greater invasiveness than empty vector-transfected cells. The stained invading cells were counted and the numbers in each group are shown in a bar graph. E Relative mRNA expression of MYO1D, KITENIN, ErbB4, GLUT1, HK2, PKM1, PKM2, and LDHA in CaCo2/EV transfected with si-PKM2. F–G Knockdown of KITENIN and MYO1D suppressed enhanced invasion capacity by PKM2. CaCo2 cells were transfected with si-KITENIN and si-MYO1D for 24 h followed by transfection of the plasmid, a construct expressing myc-tagged PKM2 (PKM2-myc) for 24 h and subjected to the transwell invasion assay. The stained invading cells were counted and the numbers in each group are shown in a bar graph. H–I Knockdown of KITENIN and MYO1D suppressed induced GLUT1, HK2, PKM2 and LDHA mRNA and protein levels by PKM2. Data are the mean ± standard deviation, n = 3 * p < 0.05; ** p < 0.01; *** p < 0.001, NS, no significant difference between groups