Fig 2: Genomic 5mC, 5hmC, 5fC, and 5caC levels in NFPAs with various SNP sites. *P < 0.05. (A) Genomic 5mC levels in the TET2 p.P29R and TET2 p.I1762V groups were lower than that in the wild-type group. (B) Genomic 5hmC level in the TET2 p.P29R group was lower than that in the wild-type group. (C) There was no significant difference of 5fC among the 7 groups. (D) There was no significant difference of 5caC among the 7 groups.

Fig 3: TET2 agonist in combination with HDACi promotes potent anti-leukemia effects in vivo. (A) Schematic showing experimental protocol. 2.5 x 106 luciferase-expressing MDS-L cells were transplanted into NSGS recipient mice intravenously. Seven days later, baseline engraftment was detected by bioluminescence imaging. Drug treatment began on D7 via I.P. injection. MDS-L engraftment was detected at D21. (B) Pseudocolored bioluminescence images of NSGS mice transplanted with MDS-L cells and subjected to indicated drug treatment. Radiance is in units of “photons/second/cm2/steradian”. One mouse in the SAHA group died during drug administration (indicated by red X). (C) Total flux (photons/sec) of NSGS mice transplanted with MDS-L cells after indicated treatments. Signal intensity was quantified from each animal shown in panel b. (D) 5hmC levels (as quantified by flow cytometry) in peripheral blood cells of mice after indicated treatments.

Fig 4: Differences in 5hmC leveland TETprotein expressionlevelbetweenPT and MT lesionsA. Representative images of H&E staining and IHC staining to show the global DNA 5hmC level and expression of TET1, TET2, and TET3 in PT and MT tissues. Scale bar, 100 µm. B. Quantitative comparison of the levels of DNA 5hmC and TET1/2/3 protein expression between PT and MT tissues. The intensities of IHC staining in invasive ductal breast cancer cells but not in mesenchymal cells were analyzed for comparison. The number of samples used for immunostaining in each group was shown in the parenthesis. Data are represented as mean ± SEM. P values are calculated by using unpaired Student’s t-test (*, P < 0.05; **, P < 0.01). Ca represents invasive ductal breast cancer cells; Me represents mesenchymal cells. H&E staining, hematoxylin and eosin staining; IHC staining, immunohistochemical staining; PT, metastasis-negative primary tumor; MT, lymph node metastasis-positive primary tumor.

Fig 5: HDAC4 is required to maintain TET2 activity. (A, B) HDAC4 was overexpressed in 293T cells and TET2 expression determined by RT-qPCR (A) or Western blot (B). (C, D) After HDAC4 knockdown using lentivirus to deliver shRNA to MDS-L cells, 5hmC levels were determined by ELISA (C) or dot blot (D). (E) hMeDIP-qPCR analysis of specific 5hmC enrichment on Chr15: 58979469-58979554, enhancer region of MTSS1 in c-kit+ cells from BM of NHD13/HDAC4 WT or NHD13/HDAC4 KO mice. Bars represent mean enrichment over input. (F) MTSS1 mRNA levels in c-kit+ cells from BM of NHD13/HDAC4 WT or NHD13/HDAC4 KO mice. (G) RT-qPCR detection of MTSS1 mRNA levels in c-kit+ cells from BM of NHD13 mice treated with SAHA or vehicle control. (H) Serial replating of NHD13/HDAC4 WT or NHD13/HDAC4 KO cells. Total BM cells from each these mice were seeded in methylcellulose medium, and colonies were counted on day 7.