Fig. 5: Surface ENO1 directly interacts with MCT4 to release lactate.

A HT29 cells were treated with RT (5 Gy) in combination with anti-ENO1 antibodies (HuL001) for 24 hr. The surface level of MCT4 was detected by flow cytometry (n = 3). WCL whole cell lysate. *p < 0.05 and ***p < 0.001. One-Way ANOVA test (n = 3). B MDA-MB-468 cells were treated with RT (5 Gy) in combination with anti-ENO1 antibodies (HuL001) for 24 hr. The surface level of MCT4 was detected by flow cytometry (n = 3). *p < 0.05 and ***p < 0.001. One-Way ANOVA test (n = 3). C HT29 cells were treated with RT (5 Gy) in combination with anti-ENO1 antibodies (HuL001) for 24 hr. The membrane fraction was isolated for immunoblotting. D MDA-MB-468 cells were treated with RT (5 Gy) in combination with anti-ENO1 antibodies (HuL001) for 24 hr. The membrane fraction was isolated for immunoblotting. E HT29 cells were transfected with HA-vector (Vec.) and HA-ENO1 for 24 hr and then treated with rhTGFβ1 protein (10 ng/mL) for 24 hr. The cell lysates were harvested for immunoprecipitation and immunoblotting. F HT29 cells were treated with rhTGFβ1 protein (10 ng/mL) for 24 hr. The colocalization of MCT4 and ENO1 was analyzed by confocal microscopy. ***p < 0.001. Unpaired t test (n = 3). G HT29 cells were individually transfected with CFP, YFP, CFP-YGP or CFP-ENO1/YFP-MCT4 for 24 hr and then treated with rhTGFβ1 protein (10 ng/mL) for 24 hr. FRET was analyzed by flow cytometry. H HT29 cells were treated with RT (5 Gy) in combination with anti-ENO1 antibodies (HuL001) for 24 hr and then analyzed by confocal microscopy. The quantification of colocalized membranous MCT4 and ENO1 is shown. *p < 0.05 and **p < 0.01. One-Way ANOVA test (n = 3).