Figure 7. TET2 is essential for SCCC numbers and survival.

(A) Expression of TET2 was evaluated by Western blot in the indicated cell lines. Transient transfection of TET2 in HEK293T cells was used as positive control. Tubulin was used as loading control. The lanes were run on the same gel but were noncontiguous. Arrowhead, TET2 protein. CTRL, control; CMV, cytomegalovirus promoter. (B–G) Analysis of SCCC and RCCC viability was evaluated in the indicated cell lines growing as MTs. (B and C) Analysis of apoptosis (B) and proportion of SCCCs (C) by flow cytometry. Data are represented as mean ± SEM of triplicates from 3 independent experiments. Blue bars, RCCCs; green bars, SCCCs. (D) Representative pictures of immunofluorescence analysis of caspase-3 (CASP3). White arrowheads, SCCCs. (E) Histological quantification of 5hmC content in RCCCs and SCCCs per picture of paraffin-embedded MTs generated from the indicated cell lines. r.u., relative units. (F) Representative pictures of double 5hmC and H2BeGFP immunostaining of paraffin-embedded TET2-WT MTs. Red arrowheads, SCCCs containing 5hmC; white arrowheads, RCCCs. (G) Representative pictures of immunofluorescence staining to detect CASP3, H2BeGFP, and 5hmC colocalization in consecutive histological sections from paraffin-embedded TET2-KO MTs. White arrowheads, apoptotic SCCCs without 5hmC content. (D, F, and G) Scale bars: 100 μm; high-magnification scale bar: 20 μm. Hoescht was used as counterstain. (B, C, and E) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, 2-tailed Student’s t test.