Fig. 6. MCL exosomes internalized by Clathrin and Caveolin1- independent endocytosis pathway.
Clathrin heavy chain (CLTC) or Caveoin1 (CAV1) down regulation. qPCR analysis of (A) CLTC and (E) CAV1 mRNA levels 72 h post electroporation of siCLTC or siCAV1 in Jeko-1 cells. Expression was normalized to housekeeping genes eIF3a and eIF3c and depicted as mRNA concentration relative to untreated cells (Mock). Data are presented as mean ± SD of thee independent experiments. (B) Representative Western Blot analysis of Clathrin heavy chain expression 72h post electroporation. α-Tubulin was used as a loading control. (C, F) PKH-67 labeled Jeko-1 exosomes were incubated with untreated (Mock), siLUC electoporated cells, siCLTC or siCAV1 treated cells, 72 h post electroporation for 60 min. Internalization was measured by flow cytometry analysis. The data are expressed as the percent of cells that uptake labeled exosomes as mean ± SD of thee independent experiments. (D) Representative confocal microscopic images of untreated (Mock), siLUC electroporated cells or siCLTC treated cells, 72 h post electroporation, incubated with PKH-26 labeled Jeko-1 exosomes or Transferin CF640 for 60 min or 15 min, respectively. Scale bar is 25μm. (G) Representative confocal microscopic images of untreated (Mock), siLUC or siCAV1 treated cells, 72 h post electroporation, incubated with PKH-26 labeled Jeko-1 exosomes or CholeraToxin-555 for 60 min. The cell membrane was labeled with AlexaFluor 488-anti CD19. Scale bar is 25μm.