Fig 2: Suppression of UHRF1 by miR-9 to 1 or shRNA causes expression upregulation of p15, p16, p21 in A549 cells. (A, B) Growth curves of miR-9 to 1, UHRF1, and EV-infected A549 (A) and NCI-H1299 (B) cells were conducted by CCK-8 assay. The OD value at 450 nm represented the viable cell numbers. (C-E) Quantitative PCR results showed the mRNA levels of p15 (C), p16 (D), and p21 (E) were induced after miR-9 to 1stable transfection of A549 cells. (F) Quantitative PCR arrays for the mRNA levels of p15 (C), p16 (D), and p21 (E) were induced after UHRF1 RNAi in A549 cells. (G, H) Western blot to quantify protein levels of p15, p16, and p21 after miR-9 to 1 stable transfection (G or UHRF1 RNAi [H] in A549 cells).

Fig 3: EBdCas9 upregulates CDKN2A (p16) expression and compromises cell viability(A) Integrative genomic viewer of CDKN2A H3K27me3 and H3K4me3 promoter tiling.(B) Timeline of EBdCas9 or NCdCas9 induction and gRNA transfection.(C) qRT-PCR analysis of EBdCas9 or NCdCas9 after 3 days of transfection with single gRNA (g1–g8) normalized to ß-actin and relative fold change compared with no guide (–g).(D) TATA boxes/initiator representation with respect to surrounding guide loci using the Element Navigation Tool for detection of core promoter elements.(E) Immunofluorescent imaging of EBdCas9 after 3 days of transfection with single p16 gRNA 1 (+g1) or no transfection (–g). Blue, DAPI; green, p16; far red, p21; scale bar, 100 µm.(F) Cell viability of EBdCas9 and NCdCas9 transfected with p16 g1 (+g1) compared with no guide (–g). Time points were analyzed every 6 h using Alamar Blue (n = 3, SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test).(G) EBdCas9 timeline for measuring epigenetic remodeling.(H) qRT-PCR of p16 relative fold change of EBdCas9 and NCdCas9 after 3 days of Dox induction and p16 g1 transfection. Samples were normalized to ß-actin and compared with no guide (–g) of each respective cell line.(I) ChIP-qPCR of induced (+Dox) EBdCas9 and NCdCas9 after 3 days of transfection with p16 g1 RNA (+g1) or no transfection (–g). Antibodies used for ChIP are listed above (Cas9, EZH2, H3K27me3). Normalized to fraction input/H3 (n = 3, SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test).(J) H3K4me3 marks increase after EBdCas9/p16 g1 (+g1) induction. CUT&RUN analysis of induced (+Dox) EBdCas9 after 3 days of transfection with p16 g1 RNA (+g1) or no transfection (–g) for H3K4me3. H3K4me3 tracks are normalized to immunoglobulin G (IgG) and displayed on the Integrated Genome Viewer (IGV); 40 kb region of the CDKN2A (p16) gene dispalyed in the integrated genome viewer.