Fig 2: Fumagillin and TNP-470 inhibit VM in HT1080 cells. (A) Treatment with the MetAP2 inhibitors fumagillin (FUM) and TNP-470 (TNP). Cells were pretreated with the inhibitors for 3 consecutive days, and then experiments were performed on day 4. (B and C) MetAP2 inhibitors suppressed VM in a dose-dependent manner. HT1080 cells were treated with FUM or TNP (0.1, 1, 10, or 100 ng/ml) and seeded on Matrigel-coated well plates. The quantification of VM formation was performed at 3 h after seeding. Scale bar, 100 µm. (D and E) MetAP2 inhibitors suppressed short-term and long-term VM. HT1080 cells were treated with 100 ng/ml FUM or TNP and seeded on Matrigel-coated well plates. The quantification of VM formation was performed at 3 and 24 h after seeding. Scale bar, 100 µm. Data shown are means ± SD. n.d., not detected. ***P<0.001. VM, vasculogenic mimicry; MetAP2, methionine aminopeptidase-2.

Fig 3: Inhibition of MetAP2 activity suppresses migration in HT1080 cells. (A and B) Cell migration was inhibited by knockout (KO) of MetAP2. Wound-healing assay in MetAP2-KO HT1080 cells was performed and the migrated areas were measured at 12 h after the scratch. Scale bar, 100 µm. (C and D) MetAP2 inhibitors suppress migration in HT1080 cells. Wound-healing assay in fumagillin (FUM)- or TNP-470 (TNP)-treated HT1080 cells was performed and the migrated areas were measured at 3 and 6 h after the scratch. Scale bar, 100 µm. Data shown are means ± SD. *P<0.05. MetAP2, methionine aminopeptidase-2.

Fig 4: MetAP2 is crucial for VM in HT1080 cells. (A) Establishment of MetAP2-knockout (KO) HT1080 cell line by CRISPR/Cas9 system. (B and C) VM was abolished by KO of MetAP2. In vitro VM formation assay in MetAP2-KO HT1080 cells was performed and the tube numbers were counted at 3 h after seeding. Empty vector (Mock) was used as a control. Scale bar, 100 µm. (D) Establishment of MetAP2-re-expressing HT1080 cell lines. (E and F) Expression of wild-type (wt) but not D251A (DA) MetAP2 rescues VM. In vitro VM formation assay in wt and DA MetAP2-re-expressing HT1080 cells was performed and the tube numbers were counted at 3 h after seeding. Empty vector (Neo) was used as a control. Scale bar, 100 µm. Data shown are means ± SD. *P<0.05, ***P<0.001. VM, vasculogenic mimicry; MetAP2, methionine aminopeptidase-2.