Fig. 2

Sensitivity to SAC abrogation in EGFR TKI resistant clones is associated with Chr22q11 amplification and upregulation of Chr22q11 genes. A Chromosome 22 WES read density maps of four representative PC9-derived clones, sensitive (S, green) and resistant (R, blue) to SAC abrogation by AURKBi and TTKi. Data were normalized using the parental cell lines. B FISH analysis of representative PC9-derived clones sensitive (S, green) and resistant (R, blue) to SAC abrogation. Probes for the three Chr22q11 genes HIRA, CRKL and MAPK1 (upper and middle panels) or for HIRA and the Chr22 telomeric gene SHANK3 (lower panels) were used. C Chr22 genes significantly up- or down-regulated by Whole Transcriptome Sequencing (WTS) in two representative PC9-derived clones with Chr22q11 amplification (A, green) or diploid (D, blue). Data were normalized against parental PC9 as explained in Supplementary Methods. D mRNA levels of four Chr22q11 genes (BCL2L13, BID, CRKL and MAPK1) in PC9 and 11-18 derived clones, classified according to Chr22q11 status; amplified (A), green vs. diploid (D), blue. RT-Q-PCR was used for mRNA quantification, data were normalized against beta-actin. A fifth gene not in Chr22 (MAPK3) was included as a control. Each point represents a clone, lines indicate mean ± SD. ****p<0.0001, ***p<0.001; ns, not significant (Student’s t test). E Western blotting analysis of four proteins coded by Chr22q11 genes in PC9 and 11-18-derived clones. B2L13 is the protein codified by the BCL2L13 gene. Erk2 (codified by MAPK1) corresponds to the lower band of the doublet and is indicated by an arrow, the upper band is Erk1 (codified by MAPK3). Green, clones with Chr22q11 amplification, sensitive to AURKBi and TTKi; blue, clones diploid for Chr22q11, resistant to both drugs. F Levels of four Chr22q11-codified proteins (B2L13, BID, CRKL and Erk1) in PC9 and 11-18 derived clones, quantified from the Western blotting image presented in (H). The intensity of the bands was normalized using β-tubulin. Clones are classified according to Chr22q11 status; A, amplified, green vs. D, diploid, blue. The Erk1 protein, not codified by a Chr22 gene, was included as a control. Each point represents a clone, lines indicate mean ± SD. ***p<0.001, **p<0.01, *p<0.05; ns, not significant (Student’s t test)