Fig. 2

CAF-induced EndoMT promotes LV abnormalities in vitro. A The morphology of HDLECs pretreated with NF/CAF-CM was observed, and untreated HDLECs served as the blank group. Cytoskeletal F-actin was stained with rhodamine-phalloidin and viewed under a fluorescence microscope at 400 × magnification (lower panel). B Western blotting analysis of VE-cadherin, α-SMA and vimentin in CM-treated HDLECs. C Transmission electron microscopy images showing the lymphatic endothelial cell–cell junction integrity after treatment with control medium, NF-CM or CAF-CM (Blue arrows indicate intercellular gap of Blank and NF-CM group; Red arrows indicate intercellular gap of CAF-CM group, panels: 40,000 × magnification; scale bar: 500 nm). D Cell proliferation was measured by CCK8 assays. n.s., not statistically significant. E Representative micrographs (left panel) of Transwell assays using HDLECs pretreated with the indicated CM are shown. Scale bar, 50 μm. The average number of migrated cells per field was calculated (right panel). F Representative micrographs (left panel) of tube formation assays using HDLECs pretreated with the indicated CM. Scale bar, 100 μm. The average tube length per field was calculated. G Confluent HDLEC monolayers were treated as indicated for 24 h. SiHa-mCherry cells were seeded onto the monolayers for another 24 h, and the number of transmigrated SiHa-mCherry cells was quantified. Data are presented as the mean ± SD of three independent experiments. *P < 0.05