Figure 6.

AC deletion suppresses cancer-initiating cell formation and self-renewal. (A) Cancer-initiating cell formation on Poly-HEMA-coated plates and vitality assays in control (scramble-treated, left) and ASAH1-null A375 cells (right). (B) Viability of cells in Poly-HEMA plates. *p < 0.05, ** p < 0,01,***p < 0.001; Student’s t test. (C) Self-renewal assay in control (scramble-treated) and ASAH1-null A375 cells: no wells were colonized by ASAH1-null cells indicating suppression of self-renewal capabilities. *p < 0.05, ** p < 0,01,***p < 0.001, Student’s t test. (C) Real-time PCR showing downregulation of SOX10, which is consistent with the observed downregulation of MITF. Two crucial genes related to cancer initiating cells formation and metabolism of xenobiotics were downregulated, CD271 and also ALDH1A3. Results are expressed as mean ± SEM, n = 3, with each experiment performed in technical and biological triplicates: experiments were performed using three replicates each and repeated three times. *p < 0.05, **p < 0,01,***p < 0.001, Student’s t test. Poly-HEMA and self-renewal experiments were performed after 30 days the CRISPR/Cas9 ablation. RNA extraction for Real-Time PCR was performed 35 days after CRISPR/Cas9 ablation.