FIGURE 3.

Generation of ΔFN1 AML12 cells. (A) mFN1 genomic structure showing the gRNA target sequence on exon 3. Also shown is the result of genomic sequencing of two knockouts. (B) Immunofluorescence detection of total FN1 with the specific antibody (in green) in WT AML12 cells but not in ΔFN1-1 or ΔFN1-2 AML12 cells. Scale bar = 50 μm. Growth curve of WT or ΔFN1 cells in (C) 10% FBS-containing medium or (D) serum-free medium. (E) Western blots showing FN1 or flotillin-1 signals in EV^WT, EV^{ΔFN1– 1}, or EV^{ΔFN1– 2} (upper two panels) and FN1 or β-actin in WT, ΔFN1-1, or ΔFN1-2 cells (lower two panels); 10 μg of proteins were loaded each lane. (F) Expression comparison of EV proteins in EV^WT or EV^{ΔFN1– 2}; 20 μg of total EV protein (approximately 2.5E + 9 particles) was loaded in each lane. PSMA6, MVP, and clusterin were identified in the proteome of EVs from AML12 cells (see Figure 1B). The experiments were repeated at least 2 times in duplicate.