Figure 4.

Quantification of the absolute number of interactions in three dimensions

(A) A typical image of the PLA-based in situ visualization of the eIF4F complex with maximum intensity projection of the spots from confocal microscope scanned z-planes. Cell outline and nuclear mask were semi-automatically determined. Scale: 5 μm.

(B) To determine the absolute number of the indicated interactions in individual cells, 20 z-planes were scanned with 0.4 μm distance between each plane and in total a 10 μm thickness of cells were acquired. Here three orthogonal axes were shown. Nuclei was counterstained with DAPI. Scale: 1 μm.

(C) Automatic pre- quantification of the spots in 3-dimensions. XY and XZ frames were shown here. Green crosses indicate the detection of the spots by the program. Scale: 5 μm.

(D) Pre-detected spots in 3- dimensions were then fitted to a gaussian model based on the intensity of each spot detected in each pixel, and the center of the spots were corrected by the gaussian fitting. This procedure largely separates those spots that localize in the same place or those that mistakenly quantified due to their diffraction between different z-planes. Scale: 5 μm.

(E) A375 melanoma cells were treated with anti-MEK reagent (Cobimetinib, Cobi) for 24 h. DMSO was used as control. The eIF4E-4EBP1 spots were visualized with PLA-based assay, individual spots were quantified by previous procedures in 3-dimensions. Scale: 5 μm.

(F) Upon MEK inhibition, an increase of the number of eIF4E-4EBP1 interactions between eIF4E-4EBP1 was observed in melanoma cells.