Journal: Cancers

Article Title: OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence

doi: 10.3390/cancers13071519

Figure Lengend Snippet: Effects of OTX015 on BRD4, MYC and GNL3 expression in OC cells. ( a ) Experiments of q-PCR (left panel) showing BRD4 mRNA levels in OTX015-exposed SKOV3 cells (0–1–3 μM), expressed as fold change relative to control cells, set at 1. β-actin mRNA was used as endogenous control. Histograms indicate mean values ± SD of two independent experiments, each performed in triplicate. Statistical significances were calculated by one-way ANOVA (* p < 0.05; ** p < 0.01). WB analysis (upper right panel) performed on nuclear (N) and cytoplasmic (C) protein extracts from OTX015-exposed SKOV3 cells (0–1–3 μM) for 72 h. Lamin B served to normalize nuclear fraction, whilst tubulin was used for the cytoplasmic fraction. IF experiments (lower right panel) showing the reduction of BRD4 nuclear staining (red) in OTX015 treated (1 μM and 3 μM) cells respect to DMSO controls. DAPI (blue) for nuclear staining. Representative images captured under ApoTome microscope at 40× magnification. ( b ) q-PCR analysis (left panel) of MYC and GNL3 expression in OTX015-exposed SKOV3 cells (0–1–3 μM), each expressed as fold change relative to mocked control cells, set at 1. β-actin mRNA was used as endogenous control. Histograms indicate mean values ± SD of two independent experiments, each performed in triplicate. Statistical significances were calculated by one-way ANOVA (* p < 0.05 vs. mocked control). WB analysis (right panel) performed on total protein extracts from SKOV3 cells treated with OTX015 (0–1–3 μM) for 72 h. Tubulin served as loading control and representative blot was shown. IF experiments (lower left panel) showing the downregulation of MYC expression and the lower intensity of GNL3 nucleolar foci in OTX015 treated (1 μM and 3 μM) cells in comparison to DMSO controls. ( c ) WB analysis of BRD4, MYC and GNL3 proteins performed on total protein extracts from SKOV3 cells transfected for 72 h with BRD4 siRNAs (si-BRD4) or negative control molecules (si-NC). Tubulin served as loading control and representative blot was shown. The original Western Blot images can be found in .

Article Snippet: SKOV3 cells (10 5 cells/well in 12-well plates) were transfected with a pool of small interfering RNAs (siRNAs) against human BRD4 (si-BRD4, sc-43639 by Santa Cruz Biotechnology), GNL3 (si-GNL3, sc-45830 by Santa Cruz Biotechnology), or siRNA negative control (si-NC, sc-37007 by Santa Cruz Biotechnology) by using RNAiMAX (Invitrogen), as previously indicated [ ].

Techniques: Expressing, Control, Staining, Microscopy, Comparison, Transfection, Negative Control, Western Blot

Journal: Cancers

Article Title: OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence

doi: 10.3390/cancers13071519

Figure Lengend Snippet: OTX015 exposure radiosensitizes OC cells by inducing apoptosis and inhibiting colony-forming ability and DNA damage repair. SKOV3 cells, treated with or without OTX015 (1 μM), were exposed or not to radiation (4 Gy). ( a ) Four h after IR, SKOV3 cells were plated at low concentration and cultured for 12 days. Representative images of colonies marked with crystal violet. Colony formation efficiency was obtained by crystal violet optical density from different assays, each in triplicate. Histograms are means ± SD. Statistical significances were calculated by two-way ANOVA: ** p < 0.01 and *** p < 0.001, vs. DMSO without IR; $$ p < 0.01 vs. DMSO/IR; ## p < 0.01 vs. OTX015 without IR. ( b ) WB of selected markers of cell cycle arrest (p21) and DNA damage/response (γH2AX, DNA-PK, ATM) in SKOV3 cells with or without OTX015 and/or IR. Tubulin served as normalizer and representative blot was shown. ( c ) WB of specific proteins involved in apoptosis (PARP, Caspase-3) were performed on SKOV3 cells treated with or without OTX015 and/or IR. Tubulin served as loading control and representative blot was shown. ( d ) q-PCR analysis (left panel) of GNL3 transcript levels in OTX015/IR-exposed SKOV3 cells, expressed as fold change over control cells (DMSO), set at 1. β-actin mRNA was used as endogenous control. Histograms indicate mean values ± SD of three independent experiments, each in triplicate. Statistical significances were calculated by two-way ANOVA (* p < 0.05 vs. DMSO; $$ p < 0.01 vs. IR). WB analysis (right panel) performed on protein extracts from OTX015/IR-exposed SKOV3 cells. Tubulin served as normalizer. The original Western Blot images can be found in .

Article Snippet: SKOV3 cells (10 5 cells/well in 12-well plates) were transfected with a pool of small interfering RNAs (siRNAs) against human BRD4 (si-BRD4, sc-43639 by Santa Cruz Biotechnology), GNL3 (si-GNL3, sc-45830 by Santa Cruz Biotechnology), or siRNA negative control (si-NC, sc-37007 by Santa Cruz Biotechnology) by using RNAiMAX (Invitrogen), as previously indicated [ ].

Techniques: Concentration Assay, Cell Culture, Control, Western Blot

Journal: Cancers

Article Title: OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence

doi: 10.3390/cancers13071519

Figure Lengend Snippet: Effects of GNL3 knocking down in OC cells. ( a ) q-PCR analysis (left panel) of GNL3 transcript levels in SKOV3 cells transfected for 72 h with GNL3 siRNAs (si-GNL3), expressed as fold change over mocked control cells (si-NC), set at 1. β-actin mRNA was used as endogenous control. WB analysis (right panel) of GNL3, γH2AX and PARP proteins performed on SKOV3 cells transfected for 72 h with GNL3 siRNAs (si-GNL3) or negative control molecules (si-NC). Tubulin was used as loading control and representative blot was shown. ( b ) Cell proliferation, evaluated by trypan blue exclusion dye in SKOV3 cells transfected with or without si-GNL3 and exposed to 1 μM OTX015, was expressed as fold change respect to si-NC/DMSO sample, set at 1. Results represent mean values ± SD of two independent experiments. Statistical significances were calculated by two-way ANOVA: * p < 0.05, ** p < 0.01; *** p < 0.001 vs. si-NC/DMSO; # p < 0.05 vs. si-GNL3/DMSO; $ p < 0.05 vs. si-NC/OTX015. ( c ) Clonogenic assays after 12 days of culture in SKOV3 cells transfected with or without si-GNL3 and treated with 1 μM OTX015. Representative images of colonies marked with crystal violet and colony formation efficiency obtained by crystal violet absorbance. Experiments were carried out twice, each in triplicate. Histograms are means ± SD. Statistical significances were calculated by two-way ANOVA: ** p < 0.01, *** p < 0.001, vs. si-NC/DMSO; # p < 0.05 vs. si-GNL3/DMSO; $ p < 0.05 vs. si-NC/OTX015. ( d ) Four h after IR, si-NC and si-GNL3 transfected SKOV3 cells were plated at low concentration and cultured for 12 days. Representative images of colonies marked with crystal violet, from two independent experiments, each performed in triplicate. Bar represents the means ± SD of the crystal violet absorbance. Statistical analyses were calculated by using two-way ANOVA: *** p < 0.001, vs. si-NC/no IR; ### p < 0.001 vs. si-GNL3/no IR; $$ p < 0.01 vs. si-NC/IR. The original Western Blot images can be found in .

Article Snippet: SKOV3 cells (10 5 cells/well in 12-well plates) were transfected with a pool of small interfering RNAs (siRNAs) against human BRD4 (si-BRD4, sc-43639 by Santa Cruz Biotechnology), GNL3 (si-GNL3, sc-45830 by Santa Cruz Biotechnology), or siRNA negative control (si-NC, sc-37007 by Santa Cruz Biotechnology) by using RNAiMAX (Invitrogen), as previously indicated [ ].

Techniques: Transfection, Control, Negative Control, Concentration Assay, Cell Culture, Western Blot

Journal: Cancers

Article Title: OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence

doi: 10.3390/cancers13071519

Figure Lengend Snippet: Biological significance of GNL3 expression in OC tumorigenesis. ( a ) GNL3 mRNA expression was assessed in 22 OC tumor biopsies and 8 non-tumoral ovarian tissues by q-PCR assays (*** p < 0.001). ( b ) Using ‘R2: Genomics Analysis and Visualization Platform ( http://r2.amc.nl (accessed on 20 December 2020)), the expression of GNL3 gene was assessed in 9 different databases referred to different Ovarian Cancers, and it results always overexpressed, n = 949, p = 2.2 × 10 −43 ( c ) The nodes acting as controller within the network are identified and classified as Hubs and BottleNecks, Hubs, or BottleNecks depending on their node degree and BottleNeck score. ( d ) A 2D Kernel Density Estimation (2D-KDE), Gaussian model, was carried out on Hubs and BottleNecks based on their node degree ( x -axis) and BottleNecks score ( y -axis).

Article Snippet: SKOV3 cells (10 5 cells/well in 12-well plates) were transfected with a pool of small interfering RNAs (siRNAs) against human BRD4 (si-BRD4, sc-43639 by Santa Cruz Biotechnology), GNL3 (si-GNL3, sc-45830 by Santa Cruz Biotechnology), or siRNA negative control (si-NC, sc-37007 by Santa Cruz Biotechnology) by using RNAiMAX (Invitrogen), as previously indicated [ ].

Techniques: Expressing