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Fig. 5 | Journal of Animal Science and Biotechnology

Fig. 5

From: An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

Fig. 5

Knocking out the NFAT5 gene with the inducible Cas9 Tet-on system. a Schematic representation of sgRNAs targeting the exonic region of the NFAT5 gene. b Western blot analysis of NFAT5 in Cas9 stable cells transfected with a single sgRNA (sgRNA-1, - 2, - 3, and - 4) or a mixture of them (sgRNA-M). Beta-tubulin served as a loading control. c The NFAT5 protein levels were quantified. The data are expressed relative to the NC group transfected with an sgRNA targeting the RFP. *P < 0.05. d NFAT5 expression in the knockout cell line. The NC transfected with sgRNA that targets the RFP and the #11 cell line transfected with sgRNA that targets NFAT5 were cultured in isotonic (300 mOsm/kg) or hypertonic (550 mOsm/kg) media for 8 h. e Schematic representation of NFAT5 gene editing in wild-type HEK293A, NC, and #11 cells. A ~ 310-nt fragment in exon 13 between the sgRNA-3 and sgRNA-4 recognition sites was inverted in #11. The premature stop codon TGA is indicated by a red arrow. f, g TauT and SMIT mRNA levels in NC and #11 cell lines under isotonic or hypertonic conditions for 8 h were measured by qRT-PCR with β-actin as an internal control. Bar charts show the relative expression level by normalization to the level of the NC group under isotonicity. Data are presented as mean ± SD (n = 3). ***P < 0.001. h Impact of NFAT5 disruption on cellular viability under hypertonicity. The NC and #11 cell lines were cultured in isotonic or hypertonic media for 24 h and subjected to a cell viability assay by the MTS method. Bar charts show the relative viability by normalization to the level of the NC group under isotonicity. Data are presented as mean ± SD (n = 6). *P < 0.05

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