Fig. 4

LncRNA LCETRL3 interacted with TDP43, prevented TDP43 degradation and activated the AKT signaling. (a) Cellular location of LCETRL3 in NSCLC PC9 and H1299 cells. (b) LCETRL3 pulldown proteins were identified by mass spectrometry in PC9 and H1299 cells. (c) Western blot validation of TDP43 pulled down by lncRNA LCETRL3. (d) LCETRL3 could be precipitated with antibody against TDP43 as compared with the IgG control in PC9 and H1299 cells. Relative enrichment (means ± SD) represents lncRNA LCETRL3 levels precipitated with TDP43 relative to an input control from three independent experiments. (e) There was markedly increased TDP43 expression in NSCLC tissues compared to normal tissues of the combined samples in the Discovery and Validation cohorts (n = 64). (f) The TDP43 protein levels in PC9 and H1299 cells after either silencing of LCETRL3 or over-expressing LCETRL3 without any treatments. (g, h) Western blot analyses of TDP43 protein levels in PC9 and H1299 cells with stable over-expressed or silenced LCETRL3 after treatment of cycloheximide (CHX). (i) Western blot analyses of the ubiquitination of TDP43 in PC9 and H1299 cells that stabilized either silenced LCETRL3 or over-expressed LCETRL3. (j, k) LCETRL3 obviously upregulated NOTCH1 expression, suppressed PTEN expression and promoted phosphorylation of AKT in PC9 cells (j) and H1299 cells (k). Two-tailed unpaired t test, ns, not significant, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001