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. 2023 Feb 22;18(1):2175522. doi: 10.1080/15592294.2023.2175522

Figure 7.

Figure 7.

Downregulation of UCHL1 by epigenetic editing suppresses the expression of COL1A1 and fibronectin. (a, b). DNA methyltransferase tools to downregulate UCHL1. HEK293T (a) and H1299 (b) cells were cotransfected with 1 µg UCHL1 sgRNA (#1/2/3) and 2 µg (in total) dCas9-mcherry-ED(s). For mCherry-based enrichment, cells were collected 60 h after transfection and dCas9-mCherry-ED expressing cells were sorted by FACS. Half of the sorted cells were harvested for short-term (day 2) expression measurements and the remaining cells were seeded in 24-well plates for long-term expression (day 12 for HEK293T, day 16 for H1299). (c, d). same as (A, B), genomic DNA at day 2 (left panel) and day 12/16 (right panel) was isolated from HEK293T (A) or H1299 (B) sorted cells, simultaneously with total RNA isolation, and used for pyrosequencing. dCas9-MSssI (E186A)-mCherry (inactive mutant); dCas9-MSssI (Q147L)-mCherry (active mutant). (e, f). H1299 cells were co-transfected with 1 µg a mixture of UCHL1 sgRNA (#1/2/3) and 2 µg dCas9-EZH2-mCherry or dCas9-NED-mCherry by PEI. Cells were collected 48 h post transfection, and mCherry-positive cells were sorted by FACS. The mRNA expression of UCHL1 (e), COL1A1, and fibronectin (f) were analysed by real-time qRT-PCR (n = 3). Significance was analysed by one-way ANOVA, *p < 0.05, **p < 0.