empty vector  (Addgene inc)

Journal: Frontiers in Oncology

Article Title: Activity of Estrogen Receptor β Agonists in Therapy-Resistant Estrogen Receptor-Positive Breast Cancer

doi: 10.3389/fonc.2022.857590

Figure Lengend Snippet: (A–C) ESR1 and ESR2 genes are differentially expressed in ERα+ parental, respective endocrine-resistant, and triple-negative breast cancer cell lines. (A, B) Expression of ESR1 and ESR2 in immortalized mammary MCF10A, transformed ERα+ MCF7 and T47D, endocrine-resistant MCF7-TamR, MCF7-FasR, T47D-TamR, and T47D-FasR, CDK6 overexpressing MCF7 (MCF7-CDK6 O/E), CDK4/6 inhibitor-resistant MCF7 (MCF7-CDK4/6iR) and T47D (T47D-CDK4/6iR), ZR-75-1, and triple-negative breast cancer (TNBC; MDA-MB231, MDA-MB-468, Hs578t) cell lines. Total RNA was isolated from the established cell lines using TRIzol. The expression of each gene was assessed by quantitative RT-PCR (qRT-PCR) performed with the DNase-treated RNA samples using gene-specific primers spanning exon–exon junctions that include large introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by the ΔΔCt method using GAPDH as an internal control. The expression of each gene is shown as the fold change relative to MCF10A. All reactions were done in triplicate, and the experiment was repeated twice. Data were plotted as mean ± SD. (A) ESR2 genes; full length (left) and all isoforms (right). (B) ESR1. (C) Whole-cell lysates were extracted, and immunoblot analyses were performed for ERβ and GAPDH (loading control) (upper panel), and ERα and GAPDH (lower panel). The intensity of the protein bands was quantified using Image Studio (LiCor) software. Numbers under the lanes of each cell line represent normalized values of the corresponding protein band (ERβ or ERα). The normalized band intensity of MCF10A was considered as 1. Immunoblot analyses were repeated twice with corresponding biological replicates. Reproducible results were obtained in each independent experiment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. For ERβ (upper panel), two different exposures were provided; low exp.= low exposure; high exp.= higher exposure of the blot. (D) ERE-luciferase-driven promoter activity upon treatment with selective ERβ agonists is significantly higher in ectopically expressing cells with ERβ compared to that of ERα. HEK293T cells were transfected with c-Flag pcDNA3 (vector control), c-Flag ERα or c-Flag ERβ in combination with ERE-Luciferase (reporter) and TK-Renilla (pRLTK; internal control) plasmids (as described in Materials and Methods ). Forty-eight hours after treatment of the cells with ERβ-specific agonists Renilla and Firefly, luciferase activities were measured using the dual-luciferase reporter assay system. Firefly luciferase was normalized to Renilla Luciferase. Treatment with: OSU-ERb-12 (0–10 µmol/l) (left) and LY500307 (0–10 µmol/l) (middle). Each assay was performed in triplicate with three experimental replicates (mean ± SD, *p < 0.05, **p < 0.01). The right panel shows equal expressions of ERα and ERβ as determined by Western blot analysis using the anti-flag antibody. Intensity of Flag-ERα/ERβ was normalized to GAPDH. The numbers under the corresponding protein band represent normalized values of the corresponding protein band intensity.

Article Snippet: Plasmids pcDNA3 (OHu23619C; pcDNA3.1 +: RRID: Addgene_10842), ERβ (OHu25562C; pcDNA3.1+ ), c-Flag pcDNA3 (OHu23619D; pcDNA3.1+/C-(K) DYK ), c-Flag ERα (OHu26586D; pcDNA3.1+/C-(K) DYK ), and c-Flag ERβ (OHu25562D; pcDNA3.1+/C-(K) DYK ) were obtained from GenScript (Piscataway, NJ, USA).

Techniques: Expressing, Transformation Assay, Isolation, Quantitative RT-PCR, Sequencing, DNA Amplification, Gene Expression, Control, Western Blot, Software, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reporter Assay

Journal: bioRxiv

Article Title: Activity of Estrogen Receptor β Agonists in Therapy-Resistant Estrogen Receptor-Positive Breast Cancer

doi: 10.1101/2022.01.14.476328

Figure Lengend Snippet: A and B , Expression of ESR1 and ESR2 in immortalized mammary MCF10A, transformed ERα+ MCF7 and T47D, endocrine resistant MCF7-TamR, MCF7-FasR, T47D-TamR, and T47D-FasR, CDK6 over-expressing MCF7 (MCF7-CDK6 O/E), CDK4/6 inhibitor resistant MCF7 (MCF7-CDK4/6iR) and T47D (T47D-CDK4/6iR), ZR-75-1, and triple negative breast cancer (TNBC; MDA-MB231, MDA-MB-468, Hs578t) cell lines. Total RNA was isolated from the established cell lines using TRIzol. The expression of each gene was assessed by quantitative RT-PCR (qRT-PCR) performed with the DNase-treated RNA samples using gene-specific primers spanning exon-exon junctions that include large introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. The expression of each gene is shown as the fold change relative to MCF10A. All reactions were done in triplicate and the experiment was repeated twice. Data were plotted as mean ± SD. A , ESR2 genes; full length (left) and all isoforms (right). B , ESR1 . C , whole-cell lysates were extracted and immunoblot analyses were performed for ERβ and GAPDH (loading control) (upper panel), and ERα and GAPDH (lower panel). Intensity of the protein bands was quantified using Image Studio (LiCor) software. Numbers under the lanes of each cell line represent normalized values of the corresponding protein band (ERβ or ERα). Normalized band intensity of MCF10A was considered as 1. Immunoblot analyses were repeated twice with corresponding biological replicates. Reproducible results were obtained in each independent experiment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. For ERβ (upper panel) two different exposures were provided; low exp.= low exposure; high exp.= higher exposure of the blot D, ERE-Luciferase driven promoter activity upon treatment with selective ERβ agonists is significantly higher in ectopically expressing cells with ERβ compared to that of ERα . HEK293T cells were transfected with c-Flag pcDNA3 (vector control), c-Flag ERα or c-Flag ERβ in combination with ERE-Luciferase (reporter) and TK-renilla (pRLTK; internal control) plasmids (as described in Materials and Methods section). Forty eight hours after treatment of the cells with ERβ specific agonists Renilla and Firefly luciferase activities were measured using the dual-luciferase reporter assay system. Renilla luciferase was normalized to Firefly luciferase. Treatment with: OSU-ERb-12 (0-10 μmol/L) (left) and LY500307 (0-10 μmol/L) (middle). Each assay was performed in triplicate with three experimental replicates. (mean + SD, *: p<0.05, **: p<0.01). Right panel shows equal expression of ERα and ERβ as determined by western blot analysis using anti-flag antibody. Intensity of Flag-ERα/ERβ was normalized to GAPDH. The numbers under the corresponding protein band represent normalized values of the corresponding protein band intensity.

Article Snippet: Plasmids expressing pcDNA3 (OHu23619C; pcDNA3.1+: RRID: Addgene_10842), ERβ (OHu25562C; pcDNA3.1+), c-Flag pcDNA3 (OHu23619D; pcDNA3.1+/C-(K) DYK), c-Flag ERα (OHu26586D; pcDNA3.1+/C-(K) DYK), and c-Flag ERβ (OHu25562D; pcDNA3.1+/C-(K) DYK were obtained from GenScript.

Techniques: Expressing, Transformation Assay, Isolation, Quantitative RT-PCR, Sequencing, DNA Amplification, Gene Expression, Control, Western Blot, Software, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reporter Assay

Journal: Arthritis Research & Therapy

Article Title: A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

doi: 10.1186/ar4379

Figure Lengend Snippet: Wnt signaling-induced upregulation of TNF-α was antagonized by DKK and Sclerostin (SOST). (A – E) Nucleus pulposus cells were transfected with the TNF-α reporter construct and increasing concentrations of the WT-DKK plasmids (100 to 500 ng) or empty backbone plasmids (A : DKK-1, B : DKK-2, C : DKK-3, D : DKK-4, E : SOST). (F - H) The TNF-α reporter plasmid was transfected into nucleus pulposus cells together with the PGL4.74 vector, and was stimulated with 6-bromoindirubin-3′-oxime (BIO) in the presence or absence of DKK expression plasmids. Luciferase activity was measured 24 h after transfection (F : DKK-3, G : DKK-4, H : SOST). * P <0.05 indicates significant differences between groups. n.s., not significant.

Article Snippet: K3-luc (TNF-α promoter element; number 11110) and the SOST expression plasmid (number 10842) were purchased from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

doi: 10.1186/ar4379

Figure Lengend Snippet: Effect of TNF-α on Wnt signaling in nucleus pulposus cells. (A - B) Cells transfected with the Topflash reporter plasmid (A) or the Fopflash plasmid (B) together with the pGL4.74 plasmid were treated with different concentrations of TNF-α for 6 to 12 h. (C) Nucleus pulposus cells were transfected with the Topflash reporter plasmid or the Fopflash reporter plasmid and increasing concentrations of the Sclerostin (SOST) plasmids (100 to 500 ng) or empty backbone plasmids. (D) SOST mRNA expression after exposure of nucleus pulposus cells to TNF-α (10 ng/mL) for 24 h, as assessed by real-time PCR. * P <0.05 indicates significant differences between groups. Error bars represent the SD. n.s., not significant.

Article Snippet: K3-luc (TNF-α promoter element; number 11110) and the SOST expression plasmid (number 10842) were purchased from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction