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. Author manuscript; available in PMC: 2017 Nov 13.

Published in final edited form as: Leukemia. 2017 May 11;31(12):2670–2677. doi: 10.1038/leu.2017.144

(a) MM.1S cells were treated with or without BG45 (20 μM) for 24 hours, further cultured with CHX (10 μg/mL), and then harvested at the indicated time intervals. The cell lysates were subjected to immunoblot analysis. (b) MM.1S cells were treated with or without BG45 (20 μM) for 24 hours; whole cell lysates were then extracted, followed by immunoprecipitation of DNMT1 or IgG as a negative control. Acetylated DNMT1 (Ac-DNMT1), ubiquitinated DNMT1 (Ub-DNMT1), and USP7 were detected by immunoblotting with Ac-K, ubiquitin, and USP7 antibody, respectively. DNMT1 served as a loading control. (c, d) 293T cells were transfected with indicated Myc-DNMT1 (WT), acetylation-dead Myc-DNMT1 (4KQ), HA-ubiquitin (Ub), Flag-USP7, and/or Flag-HDAC3 for 48 hours. In vivo ubiquitination of DNMT1 proteins was assessed by immunoprecipitation. Polyubiquitination was detected by immunoblotting with HA antibody. (b – d) As input, the whole cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH (a), DNMT1 (b), or Actin (c, d) served as a loading control. Data are representative of at least two independent experiments. (e) Schema of regulation of DNMT1 expression by HDAC3 in MM cells. HDAC3 deacetylates c-Myc, and maintains the protein stability of c-Myc. c-Myc regulates DNMT1 mRNA expression. HDAC3 deacetylates DNMT1, and maintains the protein stability of DNMT1.

(a) MM.1S cells were treated with or without BG45 (20 μM) for 24 hours, further cultured with CHX (10 μg/mL), and then harvested at the indicated time intervals. The cell lysates were subjected to immunoblot analysis. (b) MM.1S cells were treated with or without BG45 (20 μM) for 24 hours; whole cell lysates were then extracted, followed by immunoprecipitation of DNMT1 or IgG as a negative control. Acetylated DNMT1 (Ac-DNMT1), ubiquitinated DNMT1 (Ub-DNMT1), and USP7 were detected by immunoblotting with Ac-K, ubiquitin, and USP7 antibody, respectively. DNMT1 served as a loading control. (c, d) 293T cells were transfected with indicated Myc-DNMT1 (WT), acetylation-dead Myc-DNMT1 (4KQ), HA-ubiquitin (Ub), Flag-USP7, and/or Flag-HDAC3 for 48 hours. In vivo ubiquitination of DNMT1 proteins was assessed by immunoprecipitation. Polyubiquitination was detected by immunoblotting with HA antibody. (b – d) As input, the whole cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH (a), DNMT1 (b), or Actin (c, d) served as a loading control. Data are representative of at least two independent experiments. (e) Schema of regulation of DNMT1 expression by HDAC3 in MM cells. HDAC3 deacetylates c-Myc, and maintains the protein stability of c-Myc. c-Myc regulates DNMT1 mRNA expression. HDAC3 deacetylates DNMT1, and maintains the protein stability of DNMT1.