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. Author manuscript; available in PMC: 2013 Nov 20.
Published in final edited form as: Cell Rep. 2013 Oct 10;5(2):10.1016/j.celrep.2013.08.050. doi: 10.1016/j.celrep.2013.08.050

Figure 2. TET2-targeting miRNAs regulate TET2 expression and cellular 5hmC levels.

Figure 2

(A) BaF3 cells were transduced with control vector (ctrl) or indicated miRNAs, including miR-128b which does not target TET2. TET2 and HSP90 protein levels were determined by western blot. Representative data are shown out of two to three repeats. Quantification of TET2 protein level is indicated, after normalization to HSP90. (B) Quantification of the effect of indicted miRNAs on TET2 protein or RNA levels in BaF3 cells. Data were quantified from three repeats. (C) Primary mouse bone marrow cells were transduced with indicated miRNAs. Representative western blots are shown out of two (miR-125a) and three (miR-29b) experiments. (D) Genomic DNA from BaF3 cells transduced with ctrl or indicated miRNAs or a TET2 overexpression vector (TET2OE) was analyzed for 5hmC levels using dot blot assay. Blot was stained with methylene blue to control for loading. Normalized 5hmC levels are indicated. Representative blot is shown out of two to three repeats. Note that the separated 5hmC images were from the same exposure of the same blot, and same for methylene blue. (E) Quantification of 5hmC data, N=3. (F) TET2 cDNA or ctrl was co-expressed with indicated miRNAs. Cellular 5hmC was assayed with dot blot. Representative data are shown out of three repeats. (G) Quantification of data in (F). N=3. Indicated statistical significance (by *) was evaluated in comparison to Ctrl+Ctrl. (H) BaF3 cells were transduced with a control vector, or sponges that inhibit miR-29 family, miR-26 family, or miR-125 family. TET2 and HSP90 protein levels were determined by western blot. Representative data are shown from three repeats. (I) Quantification of western data in (H), N=3. (J) Cellular 5hmC levels from BaF3 cells transduced with ctrl or indicated miRNA sponges were determined by dot blot analysis. Representative data are shown from three repeats. (K) Quantification of 5hmC levels for (J), after normalizing with methylene blue. N=3. For all figures: error bars represent standard deviation. * p<0.05. See also Figures S3, S4 and Table 2.