Fig. 2

Real-time RT-PCR assays validated a subset of CRTC1-MAML2 fusion-regulated genes identified from microarray analysis. a Lentiviral pLKO.1-based shRNAs targeting various regions of the MAML2 gene were indicated. These shRNAs and scramble control shRNA (shCtl) lentiviruses were used to infect the CRTC1-MAML2 fusion-expressing H3118 MEC cells and the infected cells were processed to isolate protein lysates for Western blotting analysis and RNA for real-time RT-PCR assays. b Western blot analysis showed shM2-1 or shM2-3 led to the knockdown of MAML2 and fusion, whereas that shM2-B1 or shM2-C1 caused MAML2 knockdown only. It is noted that another shRNA, shM2-A1 targeting the exon 1 of MAML2 did not cause MAML2 knockdown. c, d Real-time RT-PCR analyses showed that knockdown of both CRTC1-MAML2 fusion and MAML2 in H3118 MEC cells led to reduced transcripts levels of a known target AREG and a subset of novel fusion target genes, including LINC00473, DMBT1, STC1, PDE4B, RUNX1, PTGS1, TGFB2, ODC1, and CDK6 (c), whereas MAML2 knockdown in H3118 MEC cells did not significantly affect their expression (d). The level of CRTC1-MAML2 fusion transcript was determined using a primer set that spans the chromosomal translocation breakpoint. The level of MAML2 knockdown was determined using the primers that amplify the exon 1 of MAML2. Data are presented as mean ± S.E. (n = 3, *p < 0.05)