Figure 6: GNG12-AS1 regulates cell migration independently of DIRAS3.

(a) Cell migration is increased in HB2, SUM159 and MCF7 cells after GNG12-AS1 depletion. Cells were treated with control, exon 1 and exon 7 GNG12-AS1 siRNAs for 48 h before the wound-healing assay was performed over 24 h time course. Cells depleted with siRNA targeted to exon 1 and exon 7 of GNG12-AS1 have significantly increased cell migration. (b) Validation of gene expression changes in SUM159 cells after depletion of GNG12-AS1 with siRNAs targeting exons 1 and 7. Expression levels of MET and MAP2K4 were measured by qRT–PCR, normalized to GAPDH and compared with control siRNA. The knockdown efficiency of GNG12-AS1 is shown in Supplementary Fig. 2b,f. (c) Immunoblot of MET, MAP2K4 and their active phosphorylated (ph) forms in SUM159 cells after depletion of GNG12-AS1 with siRNAs targeting exons 1, 2, 3 and 7. Asterisk (*) indicates a non-specific band detected with an antibody specific to phosphorylated MET. β-Tubulin was used as a loading control. For all the graphs, error bars indicate s.e.m. (n=3 biological replicates). *P<0.05, **P<0.01 and ****P<0.0001 by two-tailed Student’s t-test.