Article Title: LESR2 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C
Figure Lengend Snippet: Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-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 neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P
Article Snippet: Equal amounts of total RNA were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer’s instructions.
Techniques: Migration, In Vitro, Allele-specific Oligonucleotide, Flow Cytometry, Staining, Wound Closure Assay, Incubation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Plasmid Preparation, Infection