Fig 2: Molecular targeting of USP8 in IGROV-1 cells. (A) Quantitative RT-PCR of USP8 mRNA levels in IGROV-1 at different times after siRNA transfection; untransfected cells were used as calibrator and GAPDH as housekeeping control gene. (B) USP8 protein levels were analyzed by western blot at different times after siRNA transfection. One experiment representative of at least three is reported. Control loading is shown by vinculin. The band intensity was quantified using ImageJ, normalized to vinculin and the obtained values are reported in the Supplementary Figure S3 (C) USP8-silenced cells were seeded for apoptosis analysis by Annexin-V binding assay 48 h after transfection start and treated for 72 h with cisplatin. The columns indicate late apoptosis (Annexin V-PI posistive cells). One experiment representative of at least two is shown. (D) Twenty-four hours after transfection, cells were seeded for caspase 3/7 activation analysis by luminescent Caspase Glo asay and treated for 48 h with cisplatin. One experiment of at least two is shown. ***p < 0.0001 by One-Way ANOVA followed by Bonferroni’s test for multiple comparisons. (E) Human Apoptosis Array analysis was carried out in USP8-silenced cells with siRNA c seeded 48 h after transfection start and treated for 72 h with cisplatin. Phospho-p53 (S46) = 1,2; survivin = 3,4; claspin = 5,6. Dot intensity was quantified using ImageJ and the obtained values are reported in the Supplementary Table 2 (F) Validation of arrays by western blotting and cleaved caspase three analysis was carried out in USP8-silenced cells with siRNA b and c seeded 48 h after transfection start and treated for 72 h with cisplatin. Band intensity was quantified using ImageJ, normalized to actin/vinculin and the obtained values are reported in the Supplementary Figure S3.

Fig 3: Immunohistochemical evaluation of USP8 in ovarian carcinoma specimens. (A) Representative image of an undifferentiated case with high intensity staining (3+), showing that the protein of interest was present both at the plasma membrane and in the cytoplasm. (B) Image of a serous ovarian carcinoma with medium intensity staining (2+); (C) Image of a clear cell carcinoma with low intensity staining (1+). All samples are from stage III patients. Images showing that the protein of interest was present both at the plasma membrane and in the cytoplasm are reported. Negative control, slides incubated with secondary antibody alone.

Fig 4: Survival-related features of IGROV-1/Pt1 cells upon USP8 molecular targeting. (A) Quantitative RT-PCR of USP8 mRNA levels in IGROV-1/Pt1 at different time points after siRNA transfection; untransfected cells were the calibrator; GAPDH was used as housekeeping gene. (B) USP8 protein levels were analyzed by western blot at different time points after siRNA transfection. The reported experiment is representative of three. Vinculin was used as loading control. Quantification of the band intensity was performed with ImageJ, with normalization to vinculin and the obtained values are reported in Supplementary Figure S2 (C) Colony forming ability assayed 48 h after transfection start. Twenty-4 h after seeding IGROV-1/Pt1 cells were treated with cisplatin (continuous exposure). IC50 is the concentration inhibiting cell growth by 50% (IC50 > 1 µM for untransfected or negative control cells, IC50 = 0.78 and 0.52 µM for USP8 siRNAb and siRNAc cells, respectively). (p < 0.0001 by One-Way ANOVA followed by Bonferroni’s test for multiple comparisons). The reported values are the mean ± SD of three replicates. The reported experiment is representative of two. Representative images of cells treated with 1 μM cisplatin are shown. (D) Human phospho-RTK Array analysis 48 h after transfection start. Phospho-EGFR = 1,2; phospho-ErbB2 = 3,4; phospho-ErbB3 = 5,6. Quantification of dot intensity was performed using ImageJ. The obtained values are reported in Supplementary Table S1 (E). Western blot analysis of protein levels in USP8-silenced cells. Quantification of the band intensity was performed using ImageJ, with normalization to vinculin. The band intensities of phosphorylated Akt were referred to total forms. The obtained values are reported in the Supplementary Figure S2.

Fig 5: Molecular targeting of USP8 in PEO1 cells. (A) Quantitative RT-PCR of USP8 mRNA levels in PEO1 at different experimental time points after siRNA transfection; untransfected cells were the calibrator; GAPDH was used as housekeeping gene. (B) USP8 protein levels were analyzed by western blot at different experimental time points after siRNA transfection. The experiment is representative of three. Vinculin represents the loading control. Quantification of the band intensity was performed using ImageJ, with normalization to vinculin. The obtained values are reported in the Supplementary Figure S4 (C) Apoptosis analysis of USP8-silenced cells by Annexin-V binding assay 48 h after transfection start in cells treated for 72 h with cisplatin. The columns indicate late apoptosis (Annexin V-PI positive cells). The reported values are the mean ± SD of three technical replicates. (D) Bcl-2 and cleaved caspase 3 protein levels in USP8-silenced PEO1 cells. Forty-eight h after transfection start, cells were treated for 72 h with cisplatin and then lysed. The quantification of the band intensity was carried out using ImageJ, with normalization to actin. The obtained values are reported in the Supplementary Figure S4.