Fig. 5

The effects of ERK1 or ERK2 KO on TNFAIP3 expression. a KO of ERK1 by the CRISPR/Cas9 system in DCIS-iFGFR1 cells. A gRNA, 5’-CCACGUGCGCAAGACUCGCG, was designed based on the DNA sequence of human ERK1 gene (NM_002746.2). This gRNA should guide Cas9 to cut the position in exon 2 of the human ERK1 gene for coding the 60th amino acid (a.a.) residue. This strategy, if successful, disrupts the functions of all ERK1-splicing isoforms. Immunoblotting screening of 190 single clones identified eight KO clones, and the KO clones #2 and #5 are shown. b KO of ERK2 in DCIS-iFGFR1 cells. A gRNA, 5’-UCUUUCAUUUGCUCGAUGGU, was designed based on the human ERK2 DNA sequence (NM_002745.4). The Cas9-cutting site is corresponding to the coding sequence for the 90th a.a. residue in exon 2. This KO strategy disrupts all splicing isoforms of ERK2. Immunoblotting screening of 188 single clones identified six KO clones, and the KO clones #1 and #6 are shown. c DCIS-iFGFR1 control, ERK1 KO, and ERK2 KO cells were treated with vehicle (−) or AP20187 (+) for 24 h. TNFAIP3, p-ERK1/2, and total ERK1/2 were assayed by immunoblotting. GAPDH served as a loading control. The average ratios of TNFAIP3 band intensity to GAPDH band intensity were calculated from two independent assays. * and **p < 0.05 and p < 0.01 by one-way ANOVA