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Figure 3. Molecular targeting of USP14 in IGROV-1/Pt1 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) Colony-forming ability on plastic (left) and in soft agar (right) 48 h after transfection. For the plastic clonogenic assay, cells were continuously exposed to cisplatin and counted after 10 days. IC₅₀ is the concentration inhibiting cell growth by 50%: untransfected, 2.9 µM; negative control, 4.1 µM; USP14 siRNAa, 1.95 µM; USP14 siRNAb, 2.9 µM. For the agar assay, cells were treated with 100 µM cisplatin for 1 h, seeded in 0.33% agarose on a 0.5% agarose bed, and incubated for 2 weeks. Cell survival rates (treated versus control): 70% (untransfected), 60% (negative control), 33% (USP14 siRNAa), and 31% (USP14 siRNAb). Histograms show mean ± SD of at least three technical replicates; (D) Migratory and invasive ability of USP14-silenced cells 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. Graphs report mean ± SD of at least three technical replicates; error bars are not visible when values are nearly identical; (E) Quantitative RT-PCR of Axl mRNA levels after siRNA transfection. Untransfected cells were used as calibrator, and GAPDH served as housekeeping gene; (F) Apoptosis analysis in USP14-silenced cells. IGROV-1/Pt1 cells were seeded 48 h after transfection start and treated with cisplatin at IC₅₀ and IC₈₀ doses for 1 h, and apoptosis was assessed 24 h later by Annexin-V binding assay. Columns indicate total apoptosis. USP14: Ubiquitin-specific protease 14; RT-PCR: real-time PCR; siRNA: small interfering RNA; siRNAa: Silencer Select s17358; siRNAb: Silencer Select s17360; SD: standard deviation.