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Figure 4. Molecular targeting of USP14 in IGROV-1 cells. (A) Quantitative RT-PCR analysis of USP14 mRNA levels after siRNA transfection; untransfected cells were used as calibrator, with GAPDH as housekeeping gene; (B) Western blot analysis of USP14 protein levels at different time points after siRNA transfection, with actin as loading control; (C and D) Colony-forming ability on plastic; (C) and in soft agar (D) 48 h after transfection. Graphs report mean ± SD of at least three technical replicates. For the plastic assay, cells were continuously exposed to cisplatin and counted after 10 days. IC50 is the concentration inhibiting cell growth by 50%: untransfected, 0.27 µM; negative control, 0.36 µM; USP14 siRNAa, 0.20 µM; USP14 siRNAb, 0.19 µM. The histogram (right) shows colony numbers of untreated cells (mean ± SD, ≥ 3 technical replicates). For the agar assay, cells were treated with 3 µM cisplatin for 1 h, then seeded in 0.33% agarose on a 0.5% agarose bed, and incubated for 2 weeks. Cell survival (treated versus control): 45% (untransfected), 46% (negative control), 51% (USP14 siRNAa), and 66% (USP14 siRNAb); (E) Migratory ability of USP14-silenced cells assessed 48 h after transfection using transwell chambers in serum-free medium. After 24 h, cells were fixed in ethanol, stained with 0.4% SRB, and counted under an inverted microscope. Columns show mean ± SD of three technical replicates. USP14: Ubiquitin-specific protease 14; siRNA: small interfering RNA; siRNAa: Silencer Select s17358; siRNAb: Silencer Select s17360; SD: standard deviation.
