Fig. 2

Generation and validation of a COV434 cell line carrying a knockout of the NR2F2 gene. A Schematic representation of the human NR2F2 locus on chromosome 15. Four mRNA variants generated by alternative transcription start sites, which encode three protein isoforms, are depicted. Filled boxes indicate coding sequences (CDS), empty boxes indicate untranslated regions and lines represent introns. Arrows indicate transcriptional start sites. The position of the guide RNA (gRNA) targeting exon 2 is indicated. PAM, protospacer adjacent motif. B Genotyping of representative isolated alleles from two selected single-cell clones by Sanger sequencing revealed potential wild-type (WT) and NR2F2-knockout (KO) clones. The KO clone presented the homozygous mutation c.484delG (NM_021005), p.Gln163fs*4 (NP_066285). C Western blotting assays using total protein extracts from NT2/D1, COV434 non-transfected (NT), WT, and NR2F2-KO cell clones. The anti-COUP-TFII used recognizes the human isoform A, expressed by the NR2F2 variant 1. Alpha-tubulin was used as endogenous control. The molecular weight (kDa) is indicated. D, E RT-qPCR assay comparing relative mRNA expression of the four NR2F2 transcript variants (D) and known COUP-TFII regulated genes (E) between WT and NR2F2-KO COV434 cell clones. S8 was used as a reference gene. Values are mean ± SEM (n = 3–4). Student’s t-test with Welch’s correction, *p < 0.05, **p < 0.01, ***p < 0.001