Fig 2: Localization of the breakpoint region within the fourth intron of BPTF gene.(A) DNA samples from primary WI-38 and WI-38THP-1 were labeled and hybridized to Agilent custom arrays covering the 0.5-Mb region (the distance between probe RP11-387O17 and RP11-304I14) on chromosome 17 with an average probe spacing of 0.5 kb. (B) Quantitative Real-Time PCR analysis on genomic DNA from WI-38THP-1/2 in the BPTF gene revealed a 1.5 fold increase in BPTF genomic dosage compared to WI-38T cells. Two sets of primers were used to detect the gene dosage of BPTF. One set annealing to the 5-prime end of the BPTF gene (BPTF-exon 2) and the second set annealing to the 3-prime end of it (BPTF-exon 30).

Fig 3: Gain of the BPTF genomic locus and mRNA levels in human tumors.(A) Representative pictures of the various chromosomal rearrangements in the BPTF locus in six human tumor types. FISH analysis using probe RP11-387O17 (detecting 17q24.3 region in the BPTF locus) and a probe detecting chr17 centromeric material to distinguish between polysomy and gain of the BPTF locus. The nuclei presented are of lung adenocarcinoma cells and are representative of the different probe patterns detected in the other tumors (colon, neuroblastomas and leukimias). a) normal ploidy. b) chr17 polysomy. c) partial trisomy of 17q24.3. d) gain of 17q24.3. (B) A graph depicts the percentage of samples containing gain of 17q24.3 (a sample in which over 30% of cells exhibit gain was considered a positive sample) as assessed by FISH of a paraffin tissue array containing 143 tumor samples. (C) Expression of BPTF in several human tumors. Depicted is the relative expression of BPTF in several human cancers compared to normal tissues as obtained from the ONCOMINE database.

Fig 4: Effects of shRNA-mediated suppression of BPTF on MDA-MB-231 cells. (A) BPTF mRNA levels following shRNA-mediated suppression. (B) Mean colony number after BPTF suppression. (C) Cell cycle phases following BPTF knockdown. (D) Analysis of apoptotic rate after BPTF silencing based on detection of 7-AAD and Annexin V. (E) Western analysis of expression of various proteins following shRNA-mediated BPTF knockdown. (F) In vivo tumor cell growth following shRNA-mediated suppression of BPTF. *p < 0.05.

Fig 5: Effects of siRNA-mediated suppression of BPTF on MDA-MB-231 cells. (A) BPTF mRNA expression after siRNA knockdown. (B) Mean colony number after BPTF knockdown. (C) Cell cycle analysis following siRNA-mediated suppression of BPTF. (D) Analysis of apoptotic rate after BPTF silencing based on detection of 7-AAD and Annexin V. (E) Western analysis of expression of various proteins following siRNA-mediated BPTF knockdown. *p < 0.05.