FIGURE 3 from Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail

Enhanced in vitro cell growth and higher expression of EMT markers in GAN edited clones. A, Map of the gRNAs used in CRISPR-Cas9 assay. B, TspGW1 digested DNAs of puromycin-resistant clones of gRNAs 2 and 4 transfected ME180 cells show the presence of 287 and 155/132 bp PCR products (T and C alleles, respectively) compared with the presence of 287 bp (T/T alleles) in the parental ME180 cells. C, Representative morphology of GAN edited clones of gRNA4, C4.15.1, 15.3 in comparison with ME180 cells. D, Exon 8 PCR product sequencing shows conversion of T/T with C/C alleles in the GAN edited clone C4.15.1. Sequencing data show exon 8 SNP site containing T allele in ME180 and C allele in both the GAN edited clones C4.15.1 and C4.15.3. E, Western blot data show decreased expression of gigaxonin and e-cadherin in these clones in comparison with ME180 cells. However, there is increased expression of EMT markers, Snail, Twist, and n-cadherin in GAN edited clones. F, Incucyte growth assay shows faster growth of GAN edited clones C4.15.1 and C4.15.3 in comparison with parental ME180 cells. Cisplatin sensitivity is also reduced in these clones.