Fig 2: Clathrin relocalizes to sites of engulfment and is required for efficient completion of internalization.(A) Time course live-cell imaging of a senescent MCF-7 cell expressing CLTC-mCherry and PIK3C2B-GFP, throughout the entire process of engulfing an NIR-MCF-7 prey cell in a DOXO-NT culture. Scale bar = 100 µm. (B) Predator cell engulfment rates (upper) and confluency (lower) in DOXO-NT cultures were determined for senescent GFP-MCF-7 cells that were treated with indicated drugs or made to express an shRNA construct targeting CLTC. Concentrations of Dynasore, Filipin3, and MBCD producing toxicity are displayed as red lines (Dynasore 75 uM, 100 uM and MBCD 6 mg/mL). Underlying data can be found at S1 Data.

Fig 3: Endocytosis-related proteins are involved in the uptake of BMSC-derived sEVs by MM cells. RPMI 8226 and U266 cells were infected with lentivirus expressing shRNA against key endocytic proteins, including (A) CAV-1, (C) CLTC, (E) DNM2, and (G) Flot1 or negative control shRNA (shNC) and the expression of these proteins was determined using western blot. The pixel density of endocytosis-related proteins was quantified and normalized to GAPDH. RPMI 8226 and U266 cells infected with lentivirus expressing shRNA against key endocytic proteins, including (B) CAV-1, (D) CLTC, (F) DNM2, and (H) Flot1, or shNC were cultured with 50 µg/mL DID-labeled sEVs for 4 h and the mean fluorescence intensity of DID in these cells was determined using flow cytometry. Student's t-test was used for comparing two groups. (I-K) RPMI 8226 cells were infected with lentivirus expressing shRNA against DNM2 or CLTC or negative control shRNA (shNC) for 24 h and treated with or without bortezomib in the presence or absence of 100 µg/mL sEVs for another 48 h. (I) Their cell viability was measured using a luminescent cell viability assay. One representative result in triplicate of three experiments was presented by histograms. (J) Apoptotic cells were determined using 7-AAD and Annexin-V staining and flow cytometry. The proportions of live, early apoptotic or late apoptotic and dead cells were analyzed and presented by histograms. (K) Representative flow cytometry plots are shown. One-way ANOVA followed by multiple compressions was used for comparing multiple groups. Error bar, mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: Cltc plays an important role in exosome endocytosis. (A) qPCR analysis of expression of endocytosis-related genes (Cltc, Cav1, Pak1, and RhoA) in RAW264.7, HUVECs, and NRCMS cells, and primary hepatocytes. GAPDH served as an internal control. Data are expressed as mean ± SEM of five independent biological samples. *, p < 0.05; ns, no significance. (B) Schematic diagram of the experimental procedure. DiI-labeled exosomes were added to RAW264.7 cells treated with PBS/siControl/siClathrin to monitor exosome uptake. (C) qPCR and Western blot analysis of Cltc knockdown efficiency in RAW264.7. GAPDH served as an internal control. Data are expressed as mean ± SEM. n=5, *p < 0.05. (D) Representative fluorescence image of the DiI-labeled exosomes (red) in RAW264.7 cells. The nuclei were counter-stained with Hoechst (blue). Scale bar=5 µm. DiI-labeled exosomes (50 ng/ml) were added to RAW264.7 cells and incubated for 3 h before fluorescence microscopy.

Fig 5: Lentivirus pseudotyped with SARS-CoV-2 spike glycoprotein requires CHC for infectivity. A, HEK-293T cells stably expressing ACE2 or HEK-293T cells were incubated with lentivirus pseudotyped with the SARS-CoV-2 spike glycoprotein for 12 h. Cells were then fixed and stained with DAPI to reveal nuclei. Cells were also imaged for GFP, driven from the pseudovirus. Scale bars = 40 µm. B, HEK-293T cells stably expressing ACE2 were transfected with control siRNA or with an siRNA established to selectively knockdown the expression of CHC. The cells were incubated with lentivirus pseudotyped with the SARS-CoV-2 spike glycoprotein for 12 h. Cells were then fixed and stained with DAPI to reveal nuclei. Cells were also imaged for GFP, driven from the pseudovirus. Scale bars = 40 µm. C, the graph showing quantification of SARS-CoV-2 pseudovirus infection from experiments as in (B). n = 3 from three independent experiments, mean ± SEM; unpaired t test, *p < 0.05. ACE2, angiotensin-converting enzyme 2; CHC, clathrin heavy chain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.