Figure 5.
Figure 5. Epigenetic regulation of NRTS by DNA methylation and de novo Dnmts. (a) H9 hESCs and R1 mESCs were exposed to 1 nM AZA, 50 µM RG108, or 10 nM TSA during proliferation and differentiation. Paired-cell assays were performed on EBs 7 d after differentiation, and cell pairs were immunostained for CldU and IdU and analyzed using the double-labeling method as described in Fig. 2. (b) Quantification of the occurrence of NRTS in Dnmt-null mESCs using the CldU/IdU double-labeling method. Paired-cell assays were performed on EBs 7 d after differentiation using J1 (WT), Dnmt1-null (Dnmt1−/−), Dnmt3a-null (Dnmt3a−/−), Dnmt3b-null (Dnmt3b−/−), and Dnmt3a-Dnmt3b double-null (Dnmt3a-3b−/−) mESCs. A minimum of 100 pairs were analyzed per condition per experiment, and the percentage of cells showing asymmetric CldU (i.e., NRTS) was quantified (all cells had symmetric IdU). Data represent means ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus DMSO control (a) or versus WT mESCs (b). (c) Paired-cell assays were performed on H9-derived EBs 7 d after differentiation using the BrdU single-labeling method. Cell pairs were immunostained for BrdU and Dnmt1 (top), Dnmt3a (middle), or Dnmt3b (bottom). Representative photographs of paired cells symmetric or asymmetric for BrdU and expressing Dnmt1, Dnmt3a, or Dnmt3b are shown. (d and e) Data from experiments shown in c were quantified. (d) Paired cells symmetric for BrdU were distinguished based on Dnmt1 (top), Dnmt3a (middle), or Dnmt3b (bottom) expression pattern: symmetric and coincident with BrdU or asymmetric. (e) Paired cells asymmetric for BrdU were distinguished based on Dnmt1 (top), Dnmt3a (middle), or Dnmt3b (bottom) expression pattern: symmetric, asymmetric, and coincident with BrdU or asymmetric and mutually exclusive with BrdU. More than 100 pairs were quantified for Dnmt1, Dnmt3a, and Dnmt3b for each experiment. Data represent means ± SEM (n = 3 independent experiments). ***, P < 0.001. (f) Dnmt1, 3a, and 3b fluorescence intensity distribution among daughter cells from H9-derived EB pairs experiment. IgG background immunofluorescence was subtracted from the Dnmts immunofluorescence, and the quantification of immunofluorescence intensity of representative visually symmetric (with a distribution from equal to 60:40, as indicated) and visually asymmetric pairs of cells are shown in the scatter graph (n = 60 pairs for Dnmt1, n = 34 pairs for Dnmt3a, and n = 54 pairs for Dnmt3b representative of three independent experiments). BF, bright field.

Epigenetic regulation of NRTS by DNA methylation and de novo Dnmts. (a) H9 hESCs and R1 mESCs were exposed to 1 nM AZA, 50 µM RG108, or 10 nM TSA during proliferation and differentiation. Paired-cell assays were performed on EBs 7 d after differentiation, and cell pairs were immunostained for CldU and IdU and analyzed using the double-labeling method as described in Fig. 2. (b) Quantification of the occurrence of NRTS in Dnmt-null mESCs using the CldU/IdU double-labeling method. Paired-cell assays were performed on EBs 7 d after differentiation using J1 (WT), Dnmt1-null (Dnmt1−/−), Dnmt3a-null (Dnmt3a−/−), Dnmt3b-null (Dnmt3b−/−), and Dnmt3a-Dnmt3b double-null (Dnmt3a-3b−/−) mESCs. A minimum of 100 pairs were analyzed per condition per experiment, and the percentage of cells showing asymmetric CldU (i.e., NRTS) was quantified (all cells had symmetric IdU). Data represent means ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus DMSO control (a) or versus WT mESCs (b). (c) Paired-cell assays were performed on H9-derived EBs 7 d after differentiation using the BrdU single-labeling method. Cell pairs were immunostained for BrdU and Dnmt1 (top), Dnmt3a (middle), or Dnmt3b (bottom). Representative photographs of paired cells symmetric or asymmetric for BrdU and expressing Dnmt1, Dnmt3a, or Dnmt3b are shown. (d and e) Data from experiments shown in c were quantified. (d) Paired cells symmetric for BrdU were distinguished based on Dnmt1 (top), Dnmt3a (middle), or Dnmt3b (bottom) expression pattern: symmetric and coincident with BrdU or asymmetric. (e) Paired cells asymmetric for BrdU were distinguished based on Dnmt1 (top), Dnmt3a (middle), or Dnmt3b (bottom) expression pattern: symmetric, asymmetric, and coincident with BrdU or asymmetric and mutually exclusive with BrdU. More than 100 pairs were quantified for Dnmt1, Dnmt3a, and Dnmt3b for each experiment. Data represent means ± SEM (n = 3 independent experiments). ***, P < 0.001. (f) Dnmt1, 3a, and 3b fluorescence intensity distribution among daughter cells from H9-derived EB pairs experiment. IgG background immunofluorescence was subtracted from the Dnmts immunofluorescence, and the quantification of immunofluorescence intensity of representative visually symmetric (with a distribution from equal to 60:40, as indicated) and visually asymmetric pairs of cells are shown in the scatter graph (n = 60 pairs for Dnmt1, n = 34 pairs for Dnmt3a, and n = 54 pairs for Dnmt3b representative of three independent experiments). BF, bright field.

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