si-brd4  (Genechem)

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: 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: Oxidative Medicine and Cellular Longevity

Article Title: The Long Noncoding RNA Hotair Regulates Oxidative Stress and Cardiac Myocyte Apoptosis during Ischemia-Reperfusion Injury

doi: 10.1155/2020/1645249

Figure Lengend Snippet: Hotair expression is increased by BRD4 in response to I/R stimulation. (a) Hotair expression within the reperfused myocardium in the indicated time points ( n = 8). (b) Western blot images and the quantitative data of BRD4 in hearts subjected to ischemia for 30 minutes and reperfusion for additional 24 hours (I/R) ( n = 6). (c) Hotair expression in murine hearts after I/R injury with or without JQ1 treatment ( n = 8). (d) Hotair expression in H9c2 cells subjected to hypoxia for 2 hours and reoxygenation for the indicated time points ( n = 8). (e) Western blot images and the quantitative data of BRD4 in H9c2 cells subjected to hypoxia for 2 hours and reoxygenation for additional 24 hours (H/R) ( n = 6). (f) The efficiency of small interfering RNA against BRD4 (si Brd4 ) detected by PCR data ( n = 8). (g) Hotair expression in H9c2 cells after H/R stimulation with or without si Brd4 ( n = 8). Data are presented as mean ± standard deviation (SD). ∗ P < 0.05 versus the matched group.

Article Snippet: To notify the role of BRD4 in vitro, H9c2 cells were incubated with small interfering RNA against BRD4 (si Brd4 , 50 nM; #RSS338226, Thermo Fisher Scientific) using Lipofectamine RNAiMAX (Invitrogen) for 24 hours before H/R stimulation [ ].

Techniques: Expressing, Western Blot, Small Interfering RNA, Standard Deviation