Fig 2: Schematic diagram depicting the role of HA–DP in preserving lymphatic phenotypes. HA–DP was able to preserve key lymphatic markers, including Prox1, LYVE-1, podoplanin, and VEGFR3. When LECs are cultured on fibronectin coated plates, YAP/TAZ enter the nucleus and bind to the PROX-1 promoter, inhibiting its transcription, including its targets, such as LYVE-1, podoplanin, and VEGFR3. In contrast, culturing LECs on HA–DP coated plates enables YAP/TAZ to undergo cytoplasmic degradation, which subsequently enhance transcription of Prox1, including its targets, such as LYVE-1, podoplanin, and VEGFR3.

Fig 3: Protein and gene expression of LEC cultured on different coatings. Real-time qRT-PCR for early passage (P.5) LECs cultured on tissue culture plate (black), fibronectin-coated plate (yellow), and HA–DP (blue) coated plate for (A) LYVE-1, (B) PDPN, (C) Prox1, and (D) VEGFR3. Real-time qRT-PCR for late passage (P.7) LECs cultured on tissue culture plate (black), fibronectin-coated plate (yellow), and HA–DP (blue) coated plate for (E) LYVE-1, (F) PDPN, (G) Prox1, and (H) VEGFR3. Three biological replicates (n = 3) were collected per condition and analysed with real-time qRT-PCR with triplicate readings. Flow cytometry analysis for LECs (P.5–7) cultured on tissue culture plate (black), fibronectin-coated plate (yellow), and HA–DP (blue) coated plate indicating percentage of cells that are (I) LYVE-1+ and (J) podoplanin+. Data represents mean ± stdev., n = 4 per condition. Significance levels were set at: *P < 0.5 and **P < 0.01.

Fig 4: Immunofluorescent data show presence of key endothelial and lymphatic marker in h-iLECs. a–i represents ETV2, ETS2, and ETV2+ETS2 transduced group respectively. Blue, green, and red are indicator of DAPI, ETS-related gene and Prox1 in that order. All three-cohort shows nuclear presence of ERG and PROX1.

Fig 5: Genotypic characterization of iLECs. a–d qRT-PCR data of LYVE-1, PDPN, Prox1, and VEGRR3 of three transduced group, respectively, in each case growth factor derived LECs (endogenous activation of ETV2/ETS2) were used as control. The data show that all three transduced group showed higher expression of the genes of interest compared to control. Especially cohort having ETS2 showed very significant expression of all the LEC markers. Data represent mean ± standard deviation, n = 4 per condition. Significance levels were set at: *p < 0.5 and **p < 0.01. Three biological replicates (n = 3) were collected per condition and analyzed with real-time qRT-PCR.