Figure 5. Inhibition of rLOX-PP-Atto565 uptake in presence or absence of cytochalasin D (A – C) or LY294002 (D – F).

(A) After 3 hours rLOX-PP-Atto565 uptake was quantified by flow cytometry in absence (grey) and presence of 1.5 μM cytochalasin D (white); n = 3; *, two-tailed p-value > 0.0002. SCC9 cells were pre-incubated on ice for 30 minutes in the absence (B-1a and B-1b) or presence (B-2) of cytochalasin D. rLOX-PP-Atto565 was added in the presence or absence of cytochalasin D for an additional 15 min on ice, and then incubated at 37° C for 15 minutes in the 5% CO₂ incubator. Cells were stained for F-actin (green) and DNA (blue) and indicate that 1.5 μM cytochalasin D disrupted actin filaments, as expected. Merged Z-series images of without cytochalasin (B-1a and B1b) and with cytochalasin D (B-2) treatment were reconstructed with the LSM image viewer software. (C) The LIVE/DEAD® Fixable Near-IR stain assay determined the percentage of live cells in each sample. (NT, dark gray bars; non treated control cells; rLOX-PP-Atto565, light gray bars; rLOX-PP-Atto565 + cytochalasin D, white bars). (D) rLOX-PP-Atto565 uptake was quantified by flow cytometry in the absence (gray bar) and in the presence of 100 μM LY294002 (white bar). Data are means +/− SD; n = 3; *, two-tailed p-value>0.0006. (E) SCC9 cells were treated with rLOX-PP-Atto565 (red) for 15 minutes and stained for F-actin (green) and DNA (blue) in absence and presence of 100 μM LY294002. Merged Z-series images of B without LY294002 (above) and with LY294002 (below) treatment were reconstructed with image J software. 3 dimensional images from various angles show cup formation that open to plasma membrane (F). The LIVE/DEAD® Fixable Near-IR stain assay was employed to determine the percentage of live cells in each cell lines; NT, dark gray bars, non-treated control cells; rLOX-PP-Atto565, light gray bars; LY294002 plus rLOX-PP-Atto565, white bars.