Fig. 5

Reducing TET expression and 5hmC levels inhibits migration in a zebrafish xenograft assay. A Bright field and fluorescence examples of two larvae with accumulation of Dil stained SW480 cells within the perivitelline space and the yolk sac. The panels on the left show cells that have migrated towards the head and tail; the panels on the right show cells remaining at site of injection and no migration. B Incidence of cell migration (number embryos with foci in tail/all embryos with cells in yolk sac), after injecting SW480 cells (WT), CRISPR TET1-3 (TKO) and rescue with transiently expressed mouse Tet2 (mTet2). N = 37 (WT), 137 (TKO) and 49 (mTET2) also see Table 3 for statistics. C A representative immunoblots for 5hmC levels in WT, TKO and mTet2 rescue cells, confirming that 5hmC is reduced in TKO and rescued with transient mTet2 expression. Antibody controls include spots for nucleotide standards: 5hmC top, 5mC middle and unmodified C bottom. D Representative RT-qPCR assay confirming ectopic expression of mouse (mTeT2) in the TKO cells after transient transfection. As a control, we used a PCR assay for human (hTET2) which measures residual endogenous TET2 expression