Figure 4.

ADCs’ internalization and localization. (A) The HCT-116 cell line was seeded in imaging-specific 96-well flat-bottomed plates for 24 h to allow adherence. Then, the cells were incubated with the native Cet antibody (upper images) or ADC (lower images, Cet-IBA, Cet-RIS, or Cet-ZA) for an additional 24 h. Afterwards, cells were fixed and incubated with the anti-LAMP-1 antibody followed by staining with Alexa Fluor 488 (green color, second line of images), Alexa Fluor 647 (red color, anti-human to detect EGFR antibody, third line of images) secondary antibodies, and Sytox Orange probe to identify nuclei (blue color, first line of images). Merged images represent the overlay of the three stainings. Each image was taken at the confocal plane after scanning in sequence mode, to avoid cross-talk among the different wavelength emissions. The yellow region represents the co-localization area for the indicated markers. Bar: 100 µm, 200× magnification. (B) The HCT-116 cell line was seeded as in panel A and incubated with Cet-RIS for 24 h. Then cells were fixed and incubated with the anti-EEA-1 antibody. The yellow region, evidenced by the white arrows, indicates the overlapping area for EGFR and EEA-1. Bar: 100 µm, 200× magnification. Enlargement 10× of merge image.