Fig. 6

TNFAIP3 is required for FGFR1 activation-induced cell growth. a Generation of TNFAIP3 KO cell lines by the CRISPR/Cas9 system from DCIS-iFGFR1 cells. The gRNA 5’-UGCACCGAUACACACUGG was designed based on the human TNFAIP3 DNA sequence (NM_001270508.1) to guide Cas9 to cut the coding sequence for the a.a. residue 48 in exon 2. This targeting event disrupts the function of all three splicing variants of TNFAIP3. Immunoblotting screening of 100 individual clones identified six KO clones, and three KO clones (#3, #4 and #6) are shown. b KO of TNFAIP3 inhibited cell proliferation. DCIS-iFGFR1 #2 parent cells and TNFAIP3 KO #3 cells derived from the DCIS-iFGFR1 #2 parent cells were cultured in 6-well plate with 50,000 cells/well and treated with vehicle (DMSO) (−) or AP20187 (+) for 3 days before cells in each well were counted. Data are presented as average ± SD of six repeat assays in two independent experiments. ****p < 0.0001 by one-way ANOVA. c KO of TNFAIP3 inhibited mammosphere growth. The indicated cell lines were cultured in the U-bottom ultra-low attachment 96-well plates and treated with vehicle or AP20187 for 7 days. The DCIS-iFGFR1-#2 cell line is the parent cell line from which the TNFAIP3 KO #3 and #4 cell lines are derived. Representative images of the spheres formed were shown. The mean of the sphere diameters for each group was calculated from eight replicates. The experiment was repeated three times. ** and ***p < 0.01 and p < 0.001 by one-way ANOVA