Fig. 2

Most lncRNAs predicted to be functional showed a functional effect upon knockdown (KD). a Two-color competitive cell growth (CCG) assay. Left: K562 cells stably transduced with sgRNAs targeting a given lncRNA (or non-targeting sgRNA control)—including a blue fluorescent protein (BFP) marker gene—were mixed at a 1:1 ratio with cells transduced with sgRNA control—including a green fluorescent protein (GFP) marker gene—and the fraction of BFP-expressing cells was tracked over 14 days by flow cytometry. The effect of KD on cell proliferation was expressed as the fraction of BFP-expressing cells relative to that at day 0. Middle: a representative example of the change in the relative fraction of BFP-expressing cells throughout the experiment, normalized to day 0. Right: Representative examples of the BFP- and GFP-expressing fractions based on flow cytometry throughout the experiment. b The relative growth of BFP-expressing cells after transduction with the sgRNA control (black), thirty-nine lncRNAs predicted to be functional by INFLAMeR (green), and seven lncRNAs predicted to be non-functional (blue) at day 14 of the CCG assay. Error bars represent SD (n = 3 biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. sgRNA control using a one-tailed t-test. c Relative cell survival after 72 h incubation with 1 µM daunorubicin (DNR)—relative to that in untreated cells—for sgRNA control (black), thirty-nine lncRNAs predicted to be functional (purple), and seven lncRNAs predicted to be non-functional (blue). Error bars represent SD (n = 3 biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001 vs. sgRNA control with Bonferroni correction. (d) Of the thirty-nine predicted lncRNAs, many of those with an effect on cell proliferation (green) also affected resistance to DNR (purple) upon KD