Migration and soft agar analysis of LN229 treated with D -2-hydroxyglutarate and LN229 subclones. Migration of LN229 and subclones H3 and H114 after 72 h. LN229 cells were treated with 100 μM or 10 mM 2-HG. Subclones H3 and H114 expressed R132H protein in different amounts (A). Plotted is the open area after 24 h of incubation. Treatment with 100 μM 2-HG or ectopic expression of low amounts of R132H protein (subclone H3) increased migration speed significantly (** = p ≤ 0.01). Colony formation in soft agar of the same cells in the same setting (B). Shown are the relative colony numbers compared to the untreated parental line (100%). The 2-HG treatment could significantly (* = p ≤ 0.05) increase colony formation independent of the used concentration. An even stronger increase (** = p ≤ 0.01) could be measured for subclone H3, but no difference could be detected for subclone H114.