Fig. 2: Characterization of LRRC8A knockout HCT116 cells.

A Strategy of CRISPR/Cas9 editing applied for LRRC8A and characterization of LRRC8A KO HCT116 cells. KO-LRRC8A monoclonal HCTT16 cell lines were isolated and evaluated for the expression of their LRRC8A gene using real-time qPCR. HCT116 WT cells were used as a control, and the housekeeping genes ACTB and TBP were employed. Gene expression data were normalised to the control and presented as a percentage variation (n = 5, one-way ANOVA, ****p < 0.0001). In addition, the α-LRRC8A antibody was used to assess the protein expression of LRRC8A, with HCT116 WT cells as the control. Protein extracts (50 μg) were loaded for each sample, with actin serving as the loading control. Single clones indicated by a red asterisk were selected for further analysis. B Growth curves of KO- and WT-LRRC8A HCT116 cells. At the 96-hour, it was found that KO clones exhibited a lower proliferation rate compared to HCT116 WT control (n = 4, two-way ANOVA, ***p-value < 0.001). C Colony formation assay. LRRC8A-KO cells formed significantly fewer colonies compared to HCT116 WT control (n = 5, one-way ANOVA, *p-value < 0.5). D The Wound Healing Assay examined migratory capacity by measuring the percentage of the initial scratch area free at various time points (from t = 0 h to t = 36 h) using ImageJ software. In both experimental settings (mitomycin 5 μg/mL), HCT116 WT control and LRRC8A-KO clones displayed no significant differences in scratch closure speed (n = 4, two-way ANOVA). Illustrative histograms showing HCT116 WT and KO clone. Statistical analysis of independent triplicate experiments showed non-significant differences between WT and the KO cells in terms of apoptosis (E) and cell cycle (F). Data were compared by Nonparametric T- test with a significance level of p < 0.05.