Figure 3

CHD7 deletion inhibits anchorage-independent cell growth and spheroid invasion in LN-229 cells. (A) Strategy used to generate CHD7 KO cell clones. LN-229 cell line was co-transfected with two sgRNAs and selected with puromycin for 48 h. After confirmation of genomic editing by PCR in the mixed population selected, we performed clonal isolation. (i-ii) indicate clones carrying CHD7 mutations, which may lead to abrogation of CHD7 expression, and (iii) indicates isolated clones which still exhibit CHD7 expression. (B) Scheme indicating the sgRNA sequences targeting the 5′ and 3′ regions of the CHD7 gene. (C) CHD7 immunoblotting of nuclear extracts from two WT and two KO isolated clones. PARP1 was used as the loading control, examined in the membrane as CHD7. (D) Growth curves of LN-229 clones. 1 × 10⁴ cells were plated in 12 well plates in triplicates for each time point. Experiments were performed three times. Results are expressed as average ± SEM for three wells of a single experiment. ^*p < 0.05; one-way ANOVA followed by Dunnett’s test in comparison with WT-1. (E) 1 × 10⁴ LN-229 cells suspended in soft agar were layered onto the bottom agar in 24-well plates in triplicates. Representative images of cell colonies grown in culture medium for two weeks. Scale bar: 200 μm. The graph represents total number of colonies per well, greater than 50 μm diameter. Results are expressed as average ± SEM from three independent experiments. ^*p < 0.05, ^**p < 0.01; one-way ANOVA followed by Dunnett’s test in comparison with WT-1. (F) Spheroids of WT and KO clones were placed in a 3D-collagen I matrix and the area covered by invading cells was measured for quantification after 24, 48 and 72 h. Representative images show multicellular spheroids at the time point 0 h and 24 h. Scale bar: 400 µm. (G) Experiments were performed three times in quadruplicates for each cell clone. Results from a single representative experiment are presented. Results are expressed as average ± SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001; 2-way ANOVA followed by Bonferroni test in comparison with WT-1.