Fig 2: Expression of miRNA target mRNAs and proteins. (A) Relative expression levels of HGS and VEGFR2 in LoVo-CM-treated HUVECs relative to SW620-CM. (B) Relative expression levels of HGS and VEGFR2 in LoVo-CM-treated HUVECs relative to DMH4 treatment. The mRNA levels were normalized GAPDH, and the results are a summary of two technical repeats from three biological replicates. (C) Upper panel: western blots of HGS. Lower panel: western blots of GAPDH for loading control with for HGS. (D and E) Relative expression levels of PIK3R1 and RASA1 in (E) LoVo-CM-treated HUVECs relative to SW620-CM and (F) in LoVo-CM-treated HUVECs relative to DMH4 treatment. (F) Western blots of RASA1 and VEGFR2. Lower panel: western blots of GAPDH for loading control with for VEGFR2 and RASA1. (G and H) Graphs showing the relative expression levels of indicated protein targets, which are normalized to the GAPDH. Error bars, SEM. miRNA, miR, microRNA; HGS, hepatocyte growth factor-regulated tyrosine kinase substrate; VEGFR2, vascular endothelial growth factor receptor 2; HUVEC, human umbilical vein endothelial cell; CM, conditioned medium; RASA1, RAS P21 protein activator 1.

Fig 3: ZNRF1 mediates the recruitment of ESCRT machinery to internalized EGFR. (A–D) Wild type or ZNRF1–/– A549 cells were serum-starved overnight and stimulated with 100 ng/mL EGF for 0, 5, 10, and 20 min. (A) Cells were co-stained with EGFR (Green) and HRS (Red) antibodies. (B) Quantification of the fraction of EGFR co-localized with HRS in (A). (C) Cells were co-stained with EGFR (Green) and TSG101 (Red) antibodies. (D) Quantification of the fraction of EGFR co-localized with TSG101 in (C). (E) Cell lysates from wild type and ZNRF1–/– A549 cells were prepared and the levels of HRS and TSG101 were analyzed by immunoblotting. (F) Quantification of the size distribution of EGFR puncta in (A). White arrows indicate non-colocalized signals. Scale bar, 10 μm. Data are presented as mean ± SEM. n > 1000 puncta from at least 50 cells for each group; *P < 0.05; **P < 0.01; ***P < 0.001. All experiments were repeated two to three times with similar results.

Fig 4: ESCRT‐0 HRS interacts with ubiquitinated STING APrimary BMDMs were left untreated or treated with 50 μg/ml DMXAA for 1.5 or 3 h. Cells were lysed and a portion of lysate underwent immunoblot with the indicated antibodies (i.e. lysate). The remaining lysate was incubated with TUBE beads to isolate ubiquitinated proteins. TUBE samples underwent immunoblot with the indicated antibodies. Data are representative of two independent experiments.B Sting −/− iBMDMs expressing HA‐STING were left UT or treated with 50 μg/ml DMXAA for 1 or 3 h. Cells were lysed and a portion of lysate underwent immunoblot with the indicated antibodies (i.e. lysate). The remaining lysate underwent immunoprecipitation with an anti‐HA antibody (i.e. HA pull down). Samples then underwent immunoblot with the indicated antibodies. Data representative of three independent experiments.C Sting −/− iBMDMs expressing eGFP‐STING (yellow) were left UT or treated with 50 μg/ml DMXAA for 3 h. Cells were fixed and underwent immunofluorescence staining for conjugated ubiquitin (Ub; magenta) and HRS (cyan). Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as merged images for a single Z slice. Scale bar = 10 μm. Data shown are representative of three independent experiments.DSquare ROI indicates zoomed insert from (C), displayed as both merged and single colour images. Scale bar = 5 μm. Two colour merged images can be found in Fig EV4D.E, FThe intensity profiles were plotted for each channel for two‐line regions as indicated in (D). Data information: An additional example of STING/Ub/HRS colocalisation can be found in Appendix Fig S4. Source data are available online for this figure.

Fig 5: LAMTOR1 interacts with HRS to regulate the lysosomal degradation of PD-L1. (A) Lysates from NSCLC cells (H1975 and H358) cotransfected with LAMTOR1 or its mutants (ΔK20, ΔK31, and ΔK60) were used for immunofluorescence to demonstrate the colocalization between LAMTOR1 and LAMP1, with scale bars representing 10 μm. (B) A schematic structure of LAMTOR1 is depicted with annotations indicating WT for wild-type, ΔN for N-terminal deletion, and ΔC for C-terminal deletion. The lysates from NSCLC cells (H1975 and H358) co-transfected with GFP-LAMTOR1 mutants (ΔN1, ΔN2, ΔN3, and ΔC) and HA-HRS showed co-IP of LAMTOR1 and HRS. (C-E) Additionally, lysates from NSCLC cells (H1975 and H358) cotransfected with GFP-LAMTOR1 or its mutants (ΔN1-3, ΔN1-2, ΔN1, ΔN2, ΔN3, and ΔC) and HA-PD-L1 were subjected to Co-IP of PD-L1 and LAMTOR1. (F) Furthermore, lysates from NSCLC cells (H1975 and H358) cotransfected with GFP-LAMTOR1 mutants (WT and ΔN1) were used to analyze the co-localization of LAMP1 and PD-L1 via immunofluorescence staining, with scale bars representing 10 μm. These results are represented as mean ± SEM from six assays, with significance levels indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on Student’s t test