Fig 1: Determination of ADAR1 RNA and protein overexpression in lung cancer cell lines and its association with gene amplification. (a) Assessment of ADAR1 and ADAR2 expression by quantitative reverse-transcription PCR. Determination of ADAR1 protein levels by western blot (AMAb90535; Atlas Antibody AB, Stockholm, Sweden) (b) and immunofluorescence (ab88574; Abcam, Cambridge, UK) (c) confirms overexpression of the ADAR1 protein in NCI-H1395, NCI-H1437 and NCI-H1993 cells. (d) Fluorescence in situ hybridization of the ADAR1 gene. The UCSC genome browser (http://www.genome.ucsc.edu) was used to select the bacterial artificial chromosome (BAC) clone spanning the 1q21.3 region of the ADAR1 gene: RP11-498A2 (1q21.3–1q22). A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control. The BACs were obtained from the BACPAC Resource Center of the Children's Hospital Oakland Research Institute (Oakland, CA, USA). ADAR1 and telomeric probes were labeled with Spectrum Green and Red dUTP (Abbott, Wiesbaden, Germany), respectively, using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc., Des Plaines, IL, USA). The samples were counterstained with 4′,6-diamidino-2-phenylindole in Vectashield antifade solution (Burlingame, CA, USA). Gene amplification was observed in the interphases of NCI-H1395, NCI-H1437 and NCI-H1993 cells. Probes were verified to give a single signal on normal commercial lymphocyte metaphase slides (CGH Reagents; Abbott). (e) Assessment of ADAR1 copy number by quantitative genomic PCR. Amplification frequency of ADAR1 (evaluated with SYBR Green; Bio-Rad, Hercules, CA, USA) was calculated by the standard curve method using the 7900HT SDS program. To define an internal control gene, we chose chromosome 1p36.11 because it is the least aneuploid region among our cell lines (RPL11 gene). Primers are available upon request. DNA from the normal lung was used as the reference standard. Results are reported as n-fold copy number increase relative to the RPL11 gene. Gene amplification was observed in the NCI-H1395, NCI-H1437 and NCI-H1993 cell lines. (f) Multiplex ligation-dependent probe amplification (MLPA) assay. Two probemixes contain one probe for exons 4, 8 and 14 of the ADAR1 gene (in light blue). Twenty-one reference probes are included (in green). MLPA images from one of the two probemixes are shown. Values greater than 1 (equivalent to two copies) were considered to be extra copies. NCI-H1395, NCI-H1437 and NCI-H1993 show ADAR1 gene amplification, while A549, Cal12T and HBEC3KT-p53-K-ras are presented as examples of two ADAR1 copy number cells. (g) Graph depicting the ADAR1 amplified region in 1q21.3, identified with the Illumina Infinium HumanOmni5 microarray that interrogates 4301332 single-nucleotide polymorphisms in the entire genome. NCI-H1395 cell line has the largest amplified region (1502410 bp) that encompasses the ADAR1 gene. NCI-H1437 and NCI-H1993 have an amplified region of 767 275 and 759 573 bp, respectively. For the three cell lines there is a minimal common region of 662 085 bp where ADAR1 is included.
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