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. 2016 Nov;2(6):a001263. doi: 10.1101/mcs.a001263

Figure 2.

Figure 2.

PLAG1-ACTA2 translocation deregulates ACTA2 expression in both the lung tumor and lymph node metastases and increases lung epithelial cellular migration and invasion. (A) Schematic representation of PLAG1, ACTA2, and PLAG1-ACTA2 genomic structure with polymerase chain reaction (PCR) primer positions indicated. An exon 1 forward primer (E1FP) and an exon 3 reverse primer (E3RP) were used for PLAG1, an exon 2 forward primer (E2FP) and an exon 4 reverse primer (E4RP) were used for ACTA2, and a PLAG1 E1FP and an ACTA2 E3RP were used to PCR PLAG1-ACTA2 from cDNA. (B) Chromatogram obtained from Sanger sequencing of genomic DNA (Supplementary Fig. S) showing breakpoint region and direction of transcription. (C) Dual-color fluorescence in situ hybridization (FISH) showing PLAG1-ACTA2 gene fusion (5′PLAG1, gold; 3′ACTA2, red fluorescence signals) present in L, NSCLC (non-small-cell lung cancer), lung wedge, 2011; LN1, right cervical lymph node biopsy, 2011; LN2, right cervical lymph node biopsy, 2013; LN6 and LN7, autopsy material, 2015. (D) Gel image shows high level expression of PLAG1-ACTA2 in both lung and lymph node (right panel) when compared with expression of untranslocated PLAG2 (left panel) and ACTA2 V2 (middle panel). ACTA2 V2, PLAG1, and PLAG1-ACTA2 are not expressed in the HPL1D lung cell line control. (E) Chromatogram obtained from Sanger sequencing shows the fusion of PLAG1 exon 1 to ACTA2 exon 3 in the transcript and two potential translation initiation codons: canonical ACTA2 ATG and upstream, in-frame PLAG1 ATG. (F) Expression of PLAG1, ACTA2, and PLAG1-ACTA2 in the lung tumor, metastatic lymph nodes, and HPL1D lung cell line control were determined by quantitative reverse transcription (RT)-PCR with POLR2A mRNA as an endogenous control. Samples were analyzed in quadruplicate, and values were expressed as the mean ± SD. (G) Western blot analysis of lysates of HPL1D cells with stable expression of ACTA2 or PLAG1-ACTA2. (H) Increased chemotaxis of HPL1D cells with stable expression of ACTA2 or PLAG1-ACTA2. (I) Increased Matrigel invasion of HPL1D cells with stable expression of ACTA2 or PLAG1-ACTA2.