Fig. 4: Functional validation of coPARSE-lncRNAs.KO-rescue lentivirus plasmid construction. The plasmid contains three functional cassettes for U6 promoter-driven expression of crRNAs, Dox-inducible ectopic expression of homologs and GFP labeling for infected cells. b, IncuCyte proliferation analysis. HeLa cells maintained in a Dox-free culture medium were split into two groups (Dox+/−) for lentivirus infection, followed by transient transfection of rtTA-expression or control pcDNA3.1 plasmids 24 h after infection. GFP-positive cells were sorted by FACS for IncuCyte proliferation analysis. Error bars, means ± s.d., n = 3 biologically independent experiments. c, KO-rescue assays of 21 candidate coPARSE-lncRNAs (THORLNC as a positive control). The relative cell confluence upon Dox induction was calculated for these coPARSE-lncRNAs (the fold change of 72 h versus 0 h for each coPARSE-lncRNA was normalized to AAVS1 in the Dox+/− groups). An AAVS1-targeting crRNA pair and a segment of fly luciferase gene were used for the AAVS1 group. Error bars, means ± s.d., n = 3 biologically independent experiments, two-sided Student’s t-test. d, IncuCyte assay of the human coPARSE-lncRNA RP1-212P9.3 and its zebrafish homolog TCONS_00107744_zbf, using luciferase segments as a negative control, n = 2 biologically independent experiments. e, Time-matched images of early embryogenesis showing that injection of the four human coPARSE-lncRNAs rescued the developmental defect of the corresponding zebrafish lncRNA homolog knockdown embryos. The epiboly edge is marked by red dotted lines, and the embryonic shield is indicated by red arrowheads. Scale bars, 100 μm. f, Quantification of zebrafish lncRNA knockdown embryos complemented with human homologous coPARSE-lncRNAs, showing a rescue of the developmental delay. n = 3 biologically independent experiments. The number of embryos in each injection group is detailed in Methods. Error bars, means ± s.d., two-sided Student’s t-test. g, HeLa cell line xenograft tumors of Dox+/− groups of the human lncRNA RP1-212P9.3 KO and complementation samples by RP1-212P9.3 and its zebrafish homolog (TCONS_00107744_zbf), showing increased tumor growth of the complementation samples (top). Bar plot showing tumor weights (bottom). Error bars, means ± s.d., n = 13, 14, 6 and 7 biologically independent experiments, one-sided Student’s t-test.
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