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. 2019 Jun 28;20(13):3168. doi: 10.3390/ijms20133168

Figure 2.

Figure 2

Evaluation of UBC upregulation using the idCas9–VP64 system with sgRNAs targeting the UBC intron. (A) HEK293T cells were transiently transfected with idCas9–VP64 and scrambled sgRNA and then treated with 5 μg/mL Dox for up to 2 days. dCas9 mRNA levels were determined by qRT-PCR (n = 3 each), normalized to GAPDH, and expressed as the fold change relative to the control (0 days of Dox treatment). (B) HEK293T cells were transiently transfected with idCas9–VP64, and scrambed sgRNA (indicated as -) or sgUBC #1~#4, alone or in combination, and then treated with 5 μg/mL Dox for 2 days. dCas9–VP64 was detected by immunoblot analysis using an anti-Cas9 antibody. α-Tubulin was used as a loading control. (C) Expression of scrambled sgRNA (sgScramble) or sgUBC #1~#4 was determined by conventional RT-PCR. GAPDH was used as an internal control. (D,E) Transient transfection of HEK293T cells and Dox treatment were performed as described in (B). In (E), HEK293T cells were treated with 10 μg/mL Dox for 2 days. UBC mRNA levels were determined by qRT-PCR (n = 3 each), normalized to GAPDH, and expressed as the fold change relative to the control (-Dox, scrambled sgRNA). Representative immunoblots and RT-PCR results are shown from three independent experiments (B,C). Data are presented as means ± SEM from the indicated number of samples (A,D,E). ** p < 0.01; *** p < 0.001 vs. control (0 days of Dox treatment) or between two groups as indicated by horizontal bars. NS: Not significant.