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. 2024 Aug 28;13(17):1444. doi: 10.3390/cells13171444

Figure 2.

Figure 2

The effects of PAK4 expression on proliferation and invasiveness in osteosarcoma cells. (a,b) MTT proliferation assays (a) and colony-forming assay (b) after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. Colony-forming assays were performed after knockdown or overexpression of PAK4 in osteosarcoma cells. U2OS (3 × 103) and KHOS/NP (3 × 103) cells were seeded in culture plates for seven days. Western blot for PAK4 and GAPDH was performed to show knockdown and overexpression of PAK4 in osteosarcoma cells. (c,d) Migration (c) and invasion (d) assay after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. The migration assay was performed by seeding 5 × 104 U2OS and 5 × 104 KHOS/NP cells in the upper chamber for 48 h. The invasion assay was performed by seeding 1 × 105 U2OS and 1 × 105 KHOS/NP cells in the upper chamber for 48 h. (e) Western blotting for PAK4, FOXO3, phosphorylated FOXO3 (pFOXO3), cyclin D1, P27, BAX, BCL2, snail, TGF-β1, MMP2, and MMP9 after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells 24 h after transfection. (f) Quantitative reverse-transcription polymerase chain reaction for PAK4, FOXO3, cyclin D1, P27, BAX, BCL2, snail, TGF-β1, MMP2, and MMP9 after knockdown or overexpression of PAK4 in osteosarcoma cells. (g) Gross and histologic findings of resected tumors grown in BALB/c nude mice by implanting 1 × 106 KHOS/NP cells that were transfected with empty vectors, shRNA for PAK4, or plasmid for wild-type PAK4 into the marrow space of the right proximal tibia. The tumor volume was measured every seven days with the length × width × height × 0.52 mm3 equation. The mice were euthanized six weeks after tumor implantation. Resected tumors were H&E stained. (h) Gross and histologic findings of pulmonary metastatic nodules in BALB/c nude mice. Arrows indicate metastatic nodules. * p < 0.05; ** p < 0.001; EVs, empty vectors; PAK4-OE, vector for wild-type PAK4; shControl, control vector for shRNA; shPAK4, vector for shRNA for PAK4.