Morphology and genetic analysis of Hewga-CCS cells. (A) Phase-contrast image of Hewga-CCS cells cultured in serum-containing medium (DMEM with 10% FBS). Bar: 100 μm. (B) Phase-contrast image of Hewga-CCS cells cultured in the low-attachment plates (DMEM with 20% FBS). Bar: 100 μm. (C) Representative karyotype of Hewga-CCS. M-FISH analysis showed 5 recurrent structural chromosomal rearrangements: der(1)t(1;16), der(6)t(6;20), der(11)t(11;17), der(12)t(12;22), and der(19)t(9;19;22). (D) RT-PCR with EWS forward primer located in exon 7 and ATF1 reverse primer in exon 7 that amplifies a 779 base pair (bp) PCR product in the type 1 EWS-ATF1 transcript and a 464 bp PCR product in the type 2 EWS-ATF1 transcript. Hewga-CCS cells and the type 2 EWS-ATF1 transcript control exhibit a single band of approximately 460 bp in lane 1 and lane 4. No band is present for the negative control (NTC) of distilled water in lane 2. The type 1 EWS/ATF1 transcript control exhibits a single band of approximately 780 bp in lane 3. (E) A partial sequence chromatogram shows that ATF1 exon 5 was fused with EWS exon 7. The arrow indicates the junction of the ATF1 and EWS genes.