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. 2019 Dec 4;15(6-7):618–631. doi: 10.1080/15592294.2019.1700004

Figure 2.

Figure 2.

P16-TET induces hydroxymethylation of P16 CpG islands and reactivates expression of methylated P16 alleles in H1299 cells. (a) TAB-MSP analysis for detecting hydroxymethylated (H)- and nonhydroxymethylated (N)-P16 CpG alleles in H1299 cells stably transfected with P16-TET or empty control vector after doxycycline treatment. The MSP analysis results were also listed. Genomic DNA from RKO and BGC823 cells was used as P16M and P16U controls in the MSP assays, respectively. (b) Western blot analysis for detecting the P16 protein; Dox (±): with or without the doxycycline treatment (final conc. 0.25 μg/mL). Proteins from BGC823 cells were used as a P16U/active control. (c) qRT-PCR results for detecting P16 mRNA levels relative to Alu RNA levels; (d) Immunofluorescence confocal analysis for detecting P16 expression. P16-positive cells with P16-TET (Myc-tag) expression are highlighted with white dash cycles; P16-negative cells with P16-TET (Myc-tag) expression are highlighted with pink dash cycles. Bar: 30 μm.