Fig 2: Autophagy is involved in the clearance of accumulated EGFR.(a) The cells were pretreated with 10 mM 3-MA or 20 µM 6r for 1 h and then stimulated with 100 ng/mL EGF for additional periods of 2 and 6 h. Subsequently, the cells were harvested and subjected to SDS-PAGE and Western blotting. EGFR degradation was examined through immunostaining by using an anti-EGFR antibody. ACTIN signalling was considered the loading control. (b) After 24 h of shRNA knockdown of CTSS, the cells were treated with 100 ng/mL EGF for the indicated durations. The harvested cells were subjected to Western blotting. (c) The cells were pretreated with 20 µM 6r for 1 h and then treated with 100 ng/mL EGF for 30 min. The cells were immediately fixed, permeabilised, stained with EGFR and LC3 antibodies, as described in Methods.

Fig 3: CTSS attenuates EGF-mediated EGFR degradation.(a) OEC-M1 and MDA-MB-231 cells were pretreated with 20 µM 6r or ZFL for 1 h and subsequently incubated with 100 ng/mL EGF for an additional 2 h. The total cell lysates were analysed using EGFR-specific antibodies. ACTIN was used as the internal control for semiquantitative loading in each lane. (b) The cells were stimulated with EGF (100 ng/mL) with or without the pretreatment of 20 µM 6r for the indicated durations. EGFR degradation was examined through immunostaining by using an anti-EGFR antibody. Notably, a substantial amount of EGFR was detectable even after 6 h of EGF stimulation in 6r-treated cells. (c) The OEC-M1 cells were transiently transfected with plasmids (pCMV) that encoded wild-type CTSS. After 24 h of transfection, the cells were treated with 100 ng/mL EGF for the indicated durations and the cellular EGFR, CTSS, and ACTIN signals were determined through Western blotting. The lifespan of EGF-mediated EGFR degradation was calculated by normalising the signal intensity of EGFR with that of ACTIN. (d) The MDA-MB-231 cells were transfected with specific 50 nM CTSS siRNA (si-CTSS) for 24 h and subsequently incubated with 100 ng/mL EGF for the indicated durations. The nontargeting scramble siRNA (si-SC) was used as the scramble control. (e) The MDA-MB-231 cells were transiently transfected with plasmids encoding the CTSS-C25A mutant. After 24 h of transfection, the cells were incubated with 100 ng/mL of EGF for the indicated durations. Furthermore, the cells were harvested and subjected to SDS-PAGE and Western blotting. EGFR degradation was determined using an antibody against EGFR. ACTIN signalling was included as the loading control.

Fig 4: CTSS inhibition does not impair lysosomal activities.(a) OEC-M1 cells were pretreated with vesicle or 100 nM BAF for 1 h and then incubated with 20 µM 6r for 1 h further. Lysosomal proteolytic activities were determined using BODIPY–BSA and quantified through flow cytometry. (b) After 48 h of siRNA knockdown of CTSS and CTSB, the relative expression of CTSS and CTSB were determined through Western blotting (right panel). Lysosomal proteolytic activities were determined using BODIPY–BSA and quantified through flow cytometry (right panel). Data represent the mean ± SD of three independent experiments. Differences were found to be statistically significant at ***P < 0.001.

Fig 5: 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.