Zen system software 3

DK-MG and U-87MG ATCC cells were seeded on glass coverslips at a density of 1.0x10⁵ cells/coverslip. Upon attachment, cells were treated with 20 µg/ml PD0721-DOX ADC or DOX incubated for 1, 3 and 6 h. Subsequently, the cells were observed and images were captured using confocal laser microscope (x200 magnification; ZEN system software 3.0; Zeiss AG) and analyzed region-wide.

DK-MG and U-87MG ATCC cells were seeded onto glass coverslips at a density of 1.0x10⁵ cells/coverslip. Following attachment, cells were treated with 20 µg/ml PD0721 scFv or PD0721-DOX ADC for 2 h. Subsequently, cells were fixed with 4% paraformaldehyde (Absin Bioscience Inc.; cat no. abs9179) for 15 min in room temperature, followed by permeabilization with 0.1% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.; cat no. T8200) for 15 min. The cells were then incubated with 2% BSA (Beijing Solarbio Science & Technology Co., Ltd.; cat no. A8020) at 37˚C for 30 min, followed by incubation with FITC-H&L labelled goat anti-human IgG (Abcam; cat no. ab6854) in the dark at 37˚C for 1 h and DAPI (1:500; Absin Bioscience Inc.; cat no. abs47047616) for 5 min in room temperature. The cells were observed and images were captured with confocal laser microscope (x630 magnification; ZEN system software 3.0; Zeiss AG) and analyzed region-wide.