Figure 3.

Cellular distribution of fatty acylated EETI-II across the cytosol, plasma membrane and intracellular membranes of the ER, Golgi and mitochondria

(A) EM images of NIH3T3 cells treated with Alexa 488-conjugated peptides (50 μM for WT EETI-II; 10 μM for C12- and C14-EETI-II; 20 μM for C16-EETI-II; 40 μM for C18-EETI-II; 6 h incubation at 37°C). Cells were washed with PBS and fixed with 4% PFA in 0.1 M Sorensen phosphate buffer, pH 7.4 for 3 h at RT. Ultrathin cryosections were immunolabelled with anti-Alexa488 antibodies, detected with Protein-A-gold particles (10 nm). Samples were subjected to EM analysis as described in the STAR Methods section. Yellow shade (false colored): endosomal and lysosomal vacuoles. Circles: Alexa 488 gold particles in the cytosol. Black arrowheads: Alexa 488 gold particles associated with small membrane-lined structures, i.e. vesicles and tubules (probably endosomal vesiculo-tubular structures), or on the plasma membrane. Green arrowheads: Alexa 488 gold particles associated with mitochondria. Purple arrowheads: Alexa 488 gold particles associated with ER or Golgi. See Figure S8D for original pictures without annotations. PM: plasma membrane; L: lysosomes; E: early/late endosomes; M: mitochondrion; G: Golgi; ER: ER N: nuclei. Scale bar, 200 nm.

(B) Statistics of cytosolic distribution of the peptides. The numbers of gold particles in the “true cytosol” (cellular regions at least 20 nm away from any cellular membranes including plasma membrane, endosomes/lysosomes, small vesicles and tubules, ER, Golgi apparatus or mitochondria) were counted and divided by the “true cytosol” area.

(C) Statistics of plasma membrane (P.M.) bound peptides. The numbers of gold particles on the plasma membrane were counted and divided by P.M. area (area extending over plasma membrane and neighboring 20 nm zones on both extracellular and intracellular side, in arbitrary units). Values represent mean ± S.E.M. n > 10 images per treatment.