(A)Migration of labeled C and its 5-position modified forms by TLC under the first developing buffer. Lanes 1-3 serve as controls for the migration of 5mC and 5hmC generated from DNA oligos incubated with wild-type or catalytic mutant Tet2.
(B) The same samples used in panel A were separated by TLC under the second developing buffer. With the exception of 5mC and C, all of the other forms of C can be separated under this condition.
(C) Autoradiographs of 2D-TLC analysis of samples derived from 5mC-containing TaqI 20mer oligo DNA incubated with wild-type and catalytic-deficient mutant Tet1, Tet2, and Tet3.

(A)“X” and “Y” co-migrate with 5fC and 5caC on 2D-TLC, respectively. Left panel shows the migration pattern of the Tet2 reaction mixture on 2D-TLC. The locations of 5mC, 5hmC, “X”, and “Y” are indicated. Second panel shows the locations of control 5fC and 5caC in a parallel 2D-TLC assay. Third and fourth panels contain samples used in the first panel plus radioactive 5fC or 5caC, respectively.
(B) Confirmation of the identities of “X” and “Y” by chemical treatments. Left panel shows the migration pattern of samples derived from incubation of Tet2 with the 5mC-containing TaqI 20mer oligo DNA. Second panel demonstrates that treatment of the samples used in the left panel with NaBH₄. Third and fourth panels demonstrate that EHL and EDC respectively react with the formyl group of 5fC or the carboxyl group of 5caC to generate the new products indicated by the dotted circles.
(C) Mass spectrometric analysis demonstrates “X” has the same fingerprint as 5fC.
(D) Mass spectrometric analysis demonstrates “Y” has the same fingerprint as 5caC.

(A)Autoradiographs of 2D-TLC analysis of samples derived from 5hmC, or 5fC-containing TaqI 20mer DNA oligos incubated with wild-type or catalytic-deficient mutant Tet2.
(B) Relative percentage of 5mC, 5hmC, 5fC, and 5caC at different time points after incubation of Tet2 proteins with 5mC, 5hmC, or 5fC-containing TaqI 20mer DNA oligos.

(A)Genomic DNA prepared from either HEK293 cells or HEK293 cells expressing a GFP-tagged Tet2 were digested with TaqI, end labeled with T4 polynucleotide kinase, digested with DNase I and phosphodiesterase I, and analyzed by 2D-TLC.
(B) Mass spectrometric quantification of genomic content of 5mC, 5hmC, 5fC and 5caC relative to cytosine in HEK293 cells expressing GFP-tagged wild-type or a catalytic mutant Tet2.
(C)Mass spectrometric quantification of genomic content of 5mC, 5hmC, 5fC and 5caC relative to cytosine in mouse ES cells, Tet1 knockdown ES cells, and mouse organs. Shown are averages of two biologically independent experiments. The red dotted lines indicate the limits for accurate quantification, which are 0.8 5fC/10⁶C and 1.2 5caC/10⁶C in 20 μg of genomic DNA.