Fig. 4

Vacuolin-1 modulates a CD11b/FAK/LYN/SLP-76 signaling axis to promote RA-induced HL-60 cell differentiation. A HL-60 cells were cultured for 48 h with 1 µM RA and 0.25 µM Vacuolin-1 as indicated (Control is untreated), the cell lysates were collected, and the indicated proteins were analyzed by western blot for molecules indicated, Y416 phosphorylated SFKs, FAK, LYN, c-CBL, SLP-76. GAPDH is the loading control. B–E HL-60 cells were cultured for 48 h with 1 µM RA and 0.25 µM Vacuolin-1 as indicated, the cell lysates were collected, and FAK immunoprecipitates (IP) (B), Lyn IP (C), Slp-76 IP (D) and c-cbl IP (E) were analyzed by Western blots probed with anti-p-tyr antibody. F HL-60 cells were cultured for 48 h with 1 µM RA and 0.25 µM Vacuolin-1 as indicated, the cell lysates were collected, and the indicated proteins were analyzed by western blot, probing for the indicated molecules. G, H HL-60 cells were untreated controls or 1 µM RA-treated G or 0.25 µM Vacuolin-1 or 0.25 µM Vacuolin-1 plus 1 µM RA-treated H for 48 h as indicated, the cells were collected and stained for FAK for confocal microscopy. DAPI was used to stain nuclear DNA and delineate the nucleus