Journal: PLoS ONE

Article Title: Reactivation of Estrogen Receptor α by Vorinostat Sensitizes Mesenchymal-Like Triple-Negative Breast Cancer to Aminoflavone, a Ligand of the Aryl Hydrocarbon Receptor

doi: 10.1371/journal.pone.0074525

Figure Lengend Snippet: ( A ) Fraction-affected (Fa) versus combination index (CI) plots of sequential or simultaneous treatment with vorinostat (V) and AFP464 (AFP) in MDA-MB-231 and Hs578T cells. AFP464 and vorinostat were combined at a fixed concentration ratio of 5∶1 for both cell lines. The open triangles represent experimental CI values, and the solid lines represent simulated CI values, both of which were calculated by Calcusyn software based on the mean cell proliferation data from two independent experiments. CI >1.0, CI = 1.0, and CI <1.0 indicates antagonistic, additive, and synergistic effects, respectively. ( B ) Western blot of ERα and AhR in control and vorinostat-treated MDA-MB-231 and Hs578T cells. Cells were grown in phenol red-free medium supplemented with charcoal-stripped FBS for 1 week prior to the treatment. MDA-MB-231 and Hs578T cells were treated with vorinostat at their respective IC 50 values (2.5 and 8 µM) for 6, 12, or 24 h (Lanes 3, 4, and 5, respectively) followed by incubation in fresh medium containing E2 (100 nM) for an additional 24 h. ( C ) Immunofluorescence staining of ERα and AhR in control and vorinostat-treated MDA-MB-231 and Hs578T cells. The cells were treated in the same way as for western blot analysis. The blue DAPI, red TRITC, and green FITC staining indicates positive staining for the nucleus, AhR, and ERα, respectively. ( D ) Real-time RT-PCR determination of relative mRNA levels of ERα , CYP1A1 , and SULT1A1 in MDA-MB-231 and Hs578T cells treated with vorinostat (V) and AFP464 (AFP) at their respective IC 50 values, each alone or in sequential combination. ( E ) Real-time RT-PCR demonstrated that transient knockdown of ERα in the vorinostat-pretreated MDA-MB-231 and Hs578T cells diminished AhR-dependent transcriptional induction of CYP1A1 and SULT1A1 after AFP464 treatment. MDA-MB-231 and Hs578T cells were pretreated with vorinostat at their respective IC 50 values (2.5 or 8 µM) for 24 and 12 h, respectively, and then the cells were incubated with the ERα siRNA:transfection reagent (1∶8) mixture complexes in fresh drug-free medium for 4 h. After that, the cells were re-covered in fresh complete medium for 2 h and then treated with AFP464 at their respective IC 50 values (25 or 20 µM) for 24 h.

Article Snippet: The siRNA sequence against human ERα (catalog # sc-29305), negative control siRNA-A (sc-37007), and siRNA transfection reagent system (sc-45064) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Concentration Assay, Software, Western Blot, Incubation, Immunofluorescence, Staining, Quantitative RT-PCR, Transfection

Journal: Molecular Systems Biology

Article Title: Dissection of a complex transcriptional response using genome-wide transcriptional modelling

doi: 10.1038/msb.2009.84

Figure Lengend Snippet: Experimental inhibition of transcription factors identified by GWTM. ( A ) Electrophoretic mobility shift assay showing precipitation of DNA bound NF-κB ( * ) after irradiation (4 Gy; NT, not treated) in the presence (+) or absence (−) of the specific NF-κB inhibitor BAY 11-7082 (C, control). ( B ) Western blot showing levels of c-Jun before and after irradiation (5 Gy) in the presence or absence of transfected siRNAc-Jun. (NT, not treated; HSP90, loading control).

Article Snippet: Cells were transfected with 100 nM siRNAc-Jun (Santa Cruz Biotechnology, sc-44204) using electroporation.

Techniques: Inhibition, Electrophoretic Mobility Shift Assay, Irradiation, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Induction of Paclitaxel Resistance by ERα Mediated Prohibitin Mitochondrial-Nuclear Shuttling

doi: 10.1371/journal.pone.0083519

Figure Lengend Snippet: (A and B) ERα mediated PHB mitochondrial-nucleus translocation. PC3 cells were transfected with siRNAs specific to (A) erα or (B) erβ, or NC RNA. Twenty four hours post-transfection, 100 nM of E2 was added to the media for 96h. The PC3 cell mitochondria (M) and nuclei (N) were separated and analyzed by Western blot using PHB, ERα Histone H1 (nucleus marker), VDAC (mitochondrial marker) and Tubulin (cytoplasm marker) antibodies. T (total cell lysates). (C) ERα directly associates with PHB. 100 nM of E2 was added to the media for 96h, then PC3 cell lysates were immunoprecipitated (IP) using PHB antibody and analyzed by Western blot (WB) using the indicated antibodies (left panel). PC3 lysates were immunoprecipitated with ERα antibody, and PHB and ERα levels were analyzed by Western blot (middle panel). Equal amounts of total input PHB and ERα (Input) were used for immunoprecipitations for each condition (right). (D) ERα directly associates with PHB in mitochondria. PC3 cells were treated as in C, then the PC3 cell mitochondria were separated and immunoprecipitated as in C. (E) ERα directly associates with PHB in nucleus. PC3 cells were treated as in C, then PC3 cell nuclei were separated and immunoprecipitated as in C. Results are representative of three independent experiments.

Article Snippet: siRNAs were used corresponding to the Human ERα, ERβ and PHB genes (Santa Cruz Biotech): sierα (Cat#: sc-29305), sierβ (Cat#: sc-35325), siphb (Cat#: sc-37629). siRNAs were transfected into cells using Lipofectamine 2000.

Techniques: Translocation Assay, Transfection, Western Blot, Marker, Immunoprecipitation

Journal: Frontiers in Endocrinology

Article Title: Upregulation of an estrogen receptor-regulated gene by first generation progestins requires both the progesterone receptor and estrogen receptor alpha

doi: 10.3389/fendo.2022.959396

Figure Lengend Snippet: Both the PR and ERα are required for the upregulation of the ER-regulated gene CTSD by MPA and NET. The MCF-7 BUS cell line transfected with (A-C) 10 nM NSC or PR-A/B siRNA or (A, B, D) 25 nM NSC or ERα siRNA were treated with 100 nM E 2 , MPA or NET for 24 hours. (A) For verification of PR-A/B or ERα knockdown, total protein from the MCF-7 BUS cells transfected as described above was harvested, and western blotting performed using antibodies specific for ERα, PR-A/B and GAPDH. A representative blot is shown and (B) PR-A, PR-B and ERα expression levels were quantified relative to the GAPDH loading control using ImageJ software (Version 1.49). Western blots of three independent experiments were quantified to determine the percentage protein knocked down. (E) MCF-7 BUS cells were treated with 100 nM E 2 , MPA or NET in the absence and presence of 10 µM ICI for 24 hours. (C–E) Total RNA was isolated, reverse transcribed and real-time qPCR was conducted to determine the relative expression of CTSD mRNA levels relative to GAPDH (reference gene). The vehicle control of each condition was set as one and the relative mRNA expression in the treated samples set relative to this. Two-way ANOVA with Bonferroni’s multiple comparison post-test was used for statistical analysis. Statistically significant differences are indicated by *, ** or *** to indicate p<0.05, p<0.01 or p<0.001. No statistical significance (p>0.05) is indicated by ns.

Article Snippet: The next day the cells were transfected with either 10 nM non-silencing scrambled sequence control (NSC) siRNA (Qiagen, USA) or siRNA directed against the human PR isoforms (GS5241; a combination of 4 target-specific siRNAs, Qiagen, USA), or 25 nM NSC siRNA or siRNA directed against human ERα (SC-29305; a combination of 4 target-specific siRNAs, Santa Cruz, USA), using Dharmafect transfection reagent (Dharmacon, USA) as per the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Expressing, Software, Isolation

Journal: STAR Protocols

Article Title: Isolation of human intrahepatic leukocytes for phenotypic and functional characterization by flow cytometry

doi: 10.1016/j.xpro.2022.101356

Figure Lengend Snippet:

Article Snippet: Collagenase IV , Cell Signaling Technology , Cat# 44204S.

Techniques: Staining, Cell Counting