In vitro disruption of ZBTB20 and its effect on the WNT/CTNNB1 pathway. (A) Schematic knockout vector that carries the gRNA for targeting the BTB and zinc finger domains of ZBTB20. (B) Detection of knockout regions in genomic DNA using specific indicated primer pairs. PCR products from bands with deletion indels (dashed boxes) were extracted and sequenced. Representative PCR results from mixed and single cell cultures were shown. (C) Detection of knockout regions at the transcriptional level were also confirmed using specific primer pairs for cDNA. Representative PCR results from mixed and single cell cultures were shown. (D) Relative mRNA expression levels of AFP, CTNNB1, AXIN2, and PPARG in ZBTB20 disrupted cells. ∗∗∗∗p <0.00005; ∗∗p <0.005; ∗p <0.05. (E) Representative Western blot and protein levels of ZBTB20, total and active CTNNB1, relative to ACTB, in ZBTB20-disrupted cells. ∗∗∗∗p <0.00005; ∗∗∗p <0.0005. p, unpaired t test.