Fig. 5: Knockdown of LETR1 reduces cell growth, cell cycle progression, and migration of LECs in vitro. | Nature Communications

Fig. 5: Knockdown of LETR1 reduces cell growth, cell cycle progression, and migration of LECs in vitro.

From: LETR1 is a lymphatic endothelial-specific lncRNA governing cell proliferation and migration through KLF4 and SEMA3C

Fig. 5: Knockdown of LETR1 reduces cell growth, cell cycle progression, and migration of LECs in vitro.

a, b Cell growth profiles and cell growth rates of LECs over 48 h after ASOKD a or CRISPRi-KD b of LETR1 using IncuCyte. Confluences were normalized to T0. Growth rates were calculated as the slope of linear regression and normalized to scrambled control ASO/sgRNA. c Representative flow cytometry plots of LECs after 24 h LETR1-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). d Quantification of the cell cycle progression analysis of LECs after 24 h LETR1-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. e Representative images of the wound closure assay (9 h) in LECs after LETR1-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2 h with 2 µg/mL Mitomycin C (proliferation inhibitor) at 37 °C. Scale bar represents 200 µm. f Quantification of the wound closure assay (up to 9 h) of LECs after LETR1-ASOKD. g Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LETR1 infected LECs after 24 h LETR1-ASO2 knockdown. h Quantification of the wound closure assay (up to 9 h) of pCDH-EV and pCDH-LETR1 infected LECs after LETR1-ASO2 knockdown. Data are displayed as mean values + SD (n = 10 in a, f, and h; n = 5 in b; n = 3 in g; n = 2 in d). Percentages represent LETR1 knockdown efficiencies after the experiments. **P < 0.01, ****P < 0.0001 using ordinary one-way (for a, b, and g) and two-way (for a, b, f, and h) ANOVA with Dunnett’s multiple comparisons test against scrambled control ASO/sgRNA or LETR1-ASO2—scrambled control siRNA. In d, g, statistical analysis was performed on G0 populations. In f, h, percentages were determined for each time point using TScratch109. All displayed in vitro assays were performed in neonatal LECs derived from the same donor.

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