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Fig. 3 | Cell & Bioscience

Fig. 3

From: ZNF300 tight self-regulation and functioning through DNA methylation and histone acetylation

Fig. 3

The ZNF300 protein directly binds to ZNF300ZFR. a, b 293T cells were co-transfected with firefly luciferase reporter vector driven by SV40 promoter (a) or ZNF300 promoter (b) with ZNF300ZFR on the downstream of polyA element as described in Fig. 1d, inner control renilla luciferase reporter vector, dCas9 expressing vector, and in combination with different gRNAs specific for ZFR of ZNF300 as indicated. The promoter activity was measured, normalized, and presented as relative luciferase activity. c, d Same experiment procedure as described in a, b except that the dCas9 expressing vector was replaced with a vector expressing dCas9 fused with VP64. *, **, and *** indicate significance compared to control (p < 0.05, p < 0.01, and p < 0.001, respectively). Data (mean ± STDEV) are statistics of one representative results (triplicates) from three independent experiments with similar results. e K562 cells were transduced with control lentiviral vector (Ctrl) or a lentiviral vector expressing ZNF300. The exogenous ZNF300 protein in the resultant cells was detected by Western blot using antibody specific to Flag tag fused to exogenous ZNF300 (left panel). HSC70 serves as a loading control. The relative expression level of ZNF300 mRNA in the resultant cells was measured using quantitative RT-PCR (right panel). f K562 cells overexpressing Flag-ZNF300 were cross-linked and used for ChIP assay with Flag antibody or normal mouse IgG. The DNA amount of ZNF300ZFR and GAPDH promoter region was measured by quantitative PCR. The DNA amount was normalized to that of 1% input DNA and presented as relative enrichment. g ChIP-PCR was performed in K562 cells using antibody recognizing the endogenous ZNF300. h Wild-type (ZNF300ZFR) or mutant ZNF300ZFR (ZNF300ZFR-M) were subcloned into luciferase activity reporter vector driven by ZNF300 promoter as described in Fig. 1. i K562 cells overexpressing ZNF300 and control cells were treated with vehicle or PMA. The endogenous ZNF300 mRNA expression were measured by quantitative RT-PCR using a pair of primers recognizing the 3′ UTR that was absent in the exogenous ZNF300 expressing vector

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