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. 2020 Nov 10;48(21):12116–12134. doi: 10.1093/nar/gkaa1003

Figure 2.

Figure 2.

LSH regulates DNA methylation extending beyond its potential effect on DNA methylation by DNMT3A and DNMT3B. (A) Western blots showing knockout of both DNMT3A and DNMT3B did not affect the expression of DNMT1 and UHRF1 in HeLa cells. (B) Quantification of 5mC levels in genomic DNA derived from control and DNMT3A/3B DKO cells by HPLC. *P < 0.05, ** P< 0.005, n = 3, error bar represents SEM in all 5mC measurement. (C) Quantitative measurement of 5mC levels in genomic DNA derived from DNMT3A/3B DKO cells cultured for various days. *P < 0.05, ** P< 0.005, *** P< 0. 0005, n = 3, error bar represents SEM in all 5mC measurement. (D) Western blots validating the lack of LSH, DNMT3A/3B, and DNMT3A/3B/LSH in LSH-KO, DNMT3A/3B-DKO and DNMT3A/3B/LSH-TKO cells, respectively. (E) Quantitative measurement of 5mC levels in genomic DNA derived from LSH-KO, DNMT3A/3B-DKO and DNMT3A/3B/LSH-TKO cells. *P < 0.05, *** P< 0.0005, n = 3, error bar represents SEM in all 5mC measurement. (F) Quantitative measurement of the levels of 5mC in DNMT3A/3B/LSH-TKO cells cultured for various days. *** P< 0.0005, n = 3, error bar represents SEM in all 5mC measurement. (G) Dot-blot analysis of 5hmC in control and LSH-KO cells. Genomic DNA was prepared from each cell line and analyzed by dot-blot using anti-5hmC-specific antibody.