Fig. 6. Role of TG2 in A431 cells.

A A431 cells were grown as monolayers or in ultralow attachment plates (spheroids) in spheroid medium. After 8 d, extracts were prepared and assayed for expression of TG2 by immunoblot. Similar results were observed in four separate experiments. B A431 cells were electroporated with control- or TG2-siRNA and after 72 h cell extracts were prepared to detect TG2. C A431 cells were electroporated with the indicated siRNA and 40,000 cells were seeded into low-attachment six well dishes at time zero. Spheroid number was counted on days 1, 2 and 3. D A431 cells were seeded at 40,000 cells per six well cluster dish and after 12 h NC9 was added at time zero. Spheroid number and trypan-blue viable cell number were determined at the indicated times following NC9 addition. The values are mean ± SEM, n = 3, p < 0.05. E A431 cells, maintained as spheroids, were treated with 0 (Control) or 20 μM NC9 for 1 h and then plated atop Matrigel in 1 ml of spheroid medium in a Millicell chamber. After 24 h, the chambers were collected and stained with DAPI to detect cells that had migrated through the Matrigel to the inner surface of the membrane. The values are mean ± SEM, n = 6, p < 0.05. F A431-derived ECS cells were seeded at confluence as monolayer cultures. A wound was created and ability of the cells to close the wound was monitored with time. G Model describing regulation of spheroid formation, migration and invasion by TG2. Reduction in TG2 level, or loss of TG2 GTP binding/G-protein function, reduces ECS cell function (survival, spheroid formation, invasion and migration). NC9 inactivates TG2 TGase activity forcing it into an extended conformation to indirectly reduce GTP binding activity.