Figure 5
HMEX exosomes increase extracellular acidity of HADF. (a) HADF cells were cultured with either exosomes from HADF or HMEX for 18 h and examined for extracellular acidification rate (ECAR) during Mito Stress Test. ECAR is significantly increased in 100 μg 888-mel exosome-treated HADF compared to controls (vs HADF exosomes) (b). HMEX isolated through REIUS method reprograms metabolic profile of HADF to increase glycolysis, as demonstrated by the Glycolytic Rate Test. Rotenone and antimycin A inhibits mitochondrial respiration and the resultant ECAR increase correlates with the complete metabolic shift from OXPHOS to glycolysis. Addition of 2-deoxy-glucose (2-DG) inhibits glycolysis, resulting in ECAR decrease, confirming the ECAR produced in the experiment is due to glycolysis. HADF alone: HADF in RPMI 5% exosome depleted FBS. HADF + HADF exo: HADF cultured with their own isolated exosomes. 888-mel exo: HADF cultured with 100 μg 888-mel exosomes. (c) Basal glycolysis and compensatory glycolysis of HADF following HADF exosome or HMEX treatment, based on Glycolytic Proton Efflux Rate (glycoPER) converted from OCR and ECAR data through Seahorse XF Glycolytic Rate Assay Report. (d) L-lactate assay on HADF treated with HMEX isolated through REIUS method. d. L-lactate is synthesized as an end-product for glycolysis. Addition of HMEX from 888-mel (V600E mutant BRAF) and 2183 (wild-type BRAF) both significantly increase the amount of L-lactate synthesized by HADF over 18 h period compared to HADF exosomes added to HADF.
