Figure 3. CTSS inhibition causes EGFR accumulation in late endosomes.

(a) After the pretreatment of 20 μM 6r or ZFL for 1 h, the cells were stimulated with 100 ng/mL of EGF for the indicated durations. The cells were fixed, permeabilised, and immunostained for EGFR (green), as described in Methods. Nuclei were stained with DAPI. Note that CTSS inhibition caused EGFR accumulation in punctate intracellular vesicles. (b,c) The cells were pretreated with 20 μM 6r for 1 h and subsequently incubated with 100 ng/mL EGF for 1 h further. The cells were then homogenised and fractionated into gradients by using 10% Percoll (b) and 25% Percoll (c), as described in Methods. The Percoll gradient fractions were then subjected to SDS-PAGE, followed by Western blotting with EGFR, EEA1, Rab7, and LAMP1 antibodies. (d–f) The cells were pretreated with 20 μM 6r for 1 h and subsequently incubated with 100 ng/mL EGF for 2 h further and fixed, permeabilised, and stained with antibodies to EGFR (green; d–f) and Rab7 (red; d), EEA1 (red, e), LAMP2 (red, e), or CTSS (red; f). The cells were imaged through confocal microscopy. Scale bars, 10 μm. (d) Confocal images showed the colocalisation between accumulated EGFR and Rab7. (e) Confocal images showed that a very small amount of accumulated EGFR was colocalised with EEA1 or LAMP2. (f) Confocal images showing colocalisation between CTSS and accumulated EGFR.