Journal: Molecules

Article Title: Binding Efficacy and Thermogenic Efficiency of Pungent and Nonpungent Analogs of Capsaicin

doi: 10.3390/molecules23123198

Figure Lengend Snippet: Effect of CAP-β- D gluco, Capsiate, and CAP on mRNA levels of surrogate thermogenic markers. Mean mRNA levels ± SEM of TRPV1, SiRT-1, PRDM-16, PGC-1α, BMP8b, UCP-1, and PPARα in the inguinal WAT of NCD or HFD (±CAP, Capsiate or CAP-β- d gluco)-fed wild type mice. ** represent statistical significance for p < 0.05 for n = 4 independent experiments/condition.

Article Snippet: BMP8b , (1:100) , SC-13086; Santa Cruz Biotechnology, Inc., Dallas, TX, USA.

Techniques:

Journal: Molecules

Article Title: Binding Efficacy and Thermogenic Efficiency of Pungent and Nonpungent Analogs of Capsaicin

doi: 10.3390/molecules23123198

Figure Lengend Snippet: Effect of CAP, Capsiate, and CAP-β- d gluco on thermogenic protein. A . Representative western blots showing the expression Of TRPV1, PPARα, BMP8b, UCP-1, SiRT-1, PRDM-16, and PGC-1𝛼 in the inguinal WAT of NCD or HFD (±CAP, Capsiate or CAP-β- d gluco)-fed wild type mice. B . Mean protein intensities ± SEM for n = 3 experiments/condition. ** represent statistical significance for p < 0.05.

Article Snippet: BMP8b , (1:100) , SC-13086; Santa Cruz Biotechnology, Inc., Dallas, TX, USA.

Techniques: Western Blot, Expressing

Journal: Molecules

Article Title: Binding Efficacy and Thermogenic Efficiency of Pungent and Nonpungent Analogs of Capsaicin

doi: 10.3390/molecules23123198

Figure Lengend Snippet: Dilutions of antibodies used for immunoblotting experiments.

Article Snippet: BMP8b , (1:100) , SC-13086; Santa Cruz Biotechnology, Inc., Dallas, TX, USA.

Techniques: Western Blot

Journal: Cancers

Article Title: Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling

doi: 10.3390/cancers13092105

Figure Lengend Snippet: Association between P1 receptors, A2AR and A2BR, and clinicopathological characteristics in breast cancer patients.

Article Snippet: 4T1 and RT-R-4T1 mouse BC cells were stably transfected with expression vectors encoding shRNAs targeting A2AR (sc-39751-SH, Santa Cruz Biotechnology) or empty vectors (EV, sc-108060, Santa Cruz Biotechnology).

Techniques: Adjuvant

Journal: Cancers

Article Title: Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling

doi: 10.3390/cancers13092105

Figure Lengend Snippet: Expression levels of purinergic P2Y 2 receptor (P2Y 2 R), adenosine receptors (A1R, A2AR, A2BR, A3R), and ectonucleotidases (CD39, CD73) in various breast cancer (BC) cells and their radiotherapy-resistant (RT-R)-BC cells. Normal epithelial cell MCF-10A, triple-negative breast cancer cell (TNBC) MDA-MB231, non-TNBCs MCF-7 and T47D, and their RT-R-BC cells were treated with or without TNF-α for 24 h. Total RNA was collected from the cells, and P2Y 2 R, adenosine receptor (A1R, A2AR, A2BR, A3R), ectonucleotidases (CD39, CD73), and GAPDH mRNA expression levels were analyzed by RT-PCR as described in . Results were confirmed by repeated experiments.

Article Snippet: 4T1 and RT-R-4T1 mouse BC cells were stably transfected with expression vectors encoding shRNAs targeting A2AR (sc-39751-SH, Santa Cruz Biotechnology) or empty vectors (EV, sc-108060, Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancers

Article Title: Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling

doi: 10.3390/cancers13092105

Figure Lengend Snippet: A2AR expression is significantly increased in the tumor tissue, especially in TNBC, compared to normal epithelial tissues of breast cancer patients. ( A , B ) The expression levels of A2AR, A2BR, CD39, and CD73 were evaluated in normal epithelial tissues ( n = 20) and tumor tissues ( n = 180) ( A ) and in TNBC tissues ( n = 20) and non-TNBC tissues ( n = 160) ( B ) of patients with breast cancer. ( C ) Immunohistochemical staining of A2A2R in tumor tissues and normal epithelial tissues of breast cancer patients (scale bar, 100 μm).

Article Snippet: 4T1 and RT-R-4T1 mouse BC cells were stably transfected with expression vectors encoding shRNAs targeting A2AR (sc-39751-SH, Santa Cruz Biotechnology) or empty vectors (EV, sc-108060, Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancers

Article Title: Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling

doi: 10.3390/cancers13092105

Figure Lengend Snippet: Extracellular ADO increased cell proliferation and colony-forming ability of MDA-MB-231 and RT-R-MDA-MB-231 cells in an A2AR-dependent manner. ( A ) Cells were treated with ADO at indicated doses for 24 h, and the cell proliferation was measured by CCK-8 assay as described in . ( B ) Cells were transfected with negative control (CTRL) siRNA (100 nM) or A2AR siRNA (100 nM) for 4 h, and then the medium was replaced with fresh complete medium. After 24 h, total RNA was collected, and the efficiency of A2AR siRNA on A2AR expression was determined by RT-PCR. Then, CTRL siRNA- or A2AR siRNA-transfected (100 nM) cells were treated with ADO (100 μM) for 24 h, and then cell proliferation was measured as above. ( C,D ) CTRL siRNA- or A2AR siRNA-transfected cells were treated with ADO (100 μM) for 24 h, and then culture medium was discarded and replaced with fresh complete medium every 2–3 days. After 1–2 weeks, colony-forming ability was determined as described in . ( E,F ) Cells transfected with indicated siRNAs were treated with ADO (100 μM) for 24 h, and the invasion assay was performed and quantified as described in . The values represent the means ± SD of 5 independent experiments (scale bar, 100 μm). * p < 0.05, ** p < 0.01 compared to the control of MDA-MB-231; # p < 0.05, ## p < 0.01 compared to the ADO treatment of MDA-MB-231; §§ p < 0.01 compared to the control of RT-R-MDA-MB-231; ‡ p < 0.01 compared to the ADO treatment of RT-R-MDA-MB-231.

Article Snippet: 4T1 and RT-R-4T1 mouse BC cells were stably transfected with expression vectors encoding shRNAs targeting A2AR (sc-39751-SH, Santa Cruz Biotechnology) or empty vectors (EV, sc-108060, Santa Cruz Biotechnology).

Techniques: CCK-8 Assay, Transfection, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Invasion Assay, Control

Journal: Cancers

Article Title: Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling

doi: 10.3390/cancers13092105

Figure Lengend Snippet: Extracellular ADO activated AKT pathway and mediated β-catenin, Snail, and vimentin expression in MDA-MB-231 and RT-R-MDA-MB-231 cells. ( A,B ) Cells were time-dependently treated with ADO (100 μM), and the p-AKT, AKT ( A ), β-catenin, Snail, vimentin, and β-actin protein levels ( B ) were analyzed by Western blotting. The results were confirmed by repeated experiments. ( C – F ) Cells transfected with CTRL siRNA or A2AR siRNA were treated with ADO for 10 min (AKT), 6 h (β-catenin), 12 h (Snail), and 24 h (vimentin) for the detection of each molecule. Then, cells were harvested, and the p-AKT, AKT, β-catenin, Snail, vimentin, and β-actin protein levels in the lysates were analyzed by Western blotting, and the band intensities were assessed by scanning densitometry. The values were represented as the means ± SD of 5 independent experiments. * p < 0.05, ** p < 0.01 compared to the control of MDA-MB-231; # p < 0.05, ## p < 0.01 compared to the ADO treatment of MDA-MB-231; § p < 0.05, §§ p < 0.01 compared to the control of RT-R-MDA-MB-231; † p < 0.05, ‡ p < 0.01 compared to the ADO treatment of RT-R-MDA-MB-231.

Article Snippet: 4T1 and RT-R-4T1 mouse BC cells were stably transfected with expression vectors encoding shRNAs targeting A2AR (sc-39751-SH, Santa Cruz Biotechnology) or empty vectors (EV, sc-108060, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Cancers

Article Title: Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-β-Catenin Signaling

doi: 10.3390/cancers13092105

Figure Lengend Snippet: A2AR plays an important role in tumor growth and metastasis in in vivo mouse models. ( A ) Mouse breast cancer cells 4T1 and its RT-R-cells (RT-R-4T1) were stably transfected with empty vector (100 nM; 4T1-EV and RT-R-4T1-EV) or A2AR shRNA (100 nM; 4T1-shA2AR and RT-R-4T1-shA2AR) as described in . ( B – F ) Balb/c nude mice were divided into 4 groups ( n = 8/each group) and injected subcutaneously (1 × 10 5 cells/100 µL of serum-free medium). The animals were sacrificed on the 24th day, and the tumors and lung tissues were extracted. Tumor volumes ( B – D ) and body weights ( E ) were measured twice a week during tumor development. The incidence of lung metastasis ( F ) was investigated after sacrifice. * p < 0.05, ** p < 0.01 compared to 4T1-EV; ## p < 0.01 compared to RT-R-4T1-EV. ( G ) Tumor tissue sections were stained with anti-A2AR, anti-p-AKT, anti-β-catenin, anti-Snail, and anti-vimentin antibodies as described in (scale bar, 50 µm), and the sections were counterstained with Mayer’s hematoxylin solution. EV, empty vector; AOI, area of interest.

Article Snippet: 4T1 and RT-R-4T1 mouse BC cells were stably transfected with expression vectors encoding shRNAs targeting A2AR (sc-39751-SH, Santa Cruz Biotechnology) or empty vectors (EV, sc-108060, Santa Cruz Biotechnology).

Techniques: In Vivo, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Injection, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Marigold Supercritical Extract as Potential Co-adjuvant in Pancreatic Cancer: The Energetic Catastrophe Induced via BMP8B Ends Up With Autophagy-Induced Cell Death

doi: 10.3389/fbioe.2019.00455

Figure Lengend Snippet: BMP8B is a molecular target of marigold SFE. (A) Microarray data of differentially expressed genes after treatment of MiaPaCa-2 pancreatic cancer cell line with 30 and 70 μg/ml marigold SFE for 48 h. Data represent the value of the most significant probe for three independent experiments for each condition. Genes with a statistically significant difference ( P value < 0.05) and more than two-fold absolute change variation compared to control (DMSO) are shown. (B) Validation of the upregulation of BMP8B after marigold SFE treatment in two pancreatic cancer cell lines, MiaPaca-2 and Panc-1. Non-treated cells were kept as controls. Data represent the mean ± S.E.M. of three independent experiments, each one performed in triplicate. Asterisks indicate statistical differences in treated cells in comparison with control (DMSO). ** P < 0.05, **** P < 0.001 indicate statistically significant differences refered to the control C.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was performed in the 7900HT Real-Time PCR System (Life Technologies) using the VeriQuest SYBR Green qPCR Master Mix (Affymetrix, Santa Clara, CA, USA), and Taqman probes were used: Hs01629120_s1, Hs01029413_m1, Hs00245183_m1, and Hs99999901_s1 for BMP8B, TFAP2A, ZFP36L1 , and 18S, respectively; or oligos in the case of the epithelial-to-mesenchymal transition (EMT), stemness and endoplasmic reticulum (ER) stress markers ( displays the list and sequences of the primers used).

Techniques: Microarray

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Marigold Supercritical Extract as Potential Co-adjuvant in Pancreatic Cancer: The Energetic Catastrophe Induced via BMP8B Ends Up With Autophagy-Induced Cell Death

doi: 10.3389/fbioe.2019.00455

Figure Lengend Snippet: Depletion of BMP8B counteracts the effect of marigold SFE on the inhibition of mitochondrial oxidative phosphorylation. (A) Mitochondrial respiration analysis by flux analysis of the OCR in MiaPaca-2 transfected with si-RNA pools against BMP8B or si-RNA-scramble, in non-treated cells and marigold SFE-treated cells (1 × IC 50 , 2 × IC 50 ). Lower panels: basal respiration rate, spare respiratory capacity, ATP production, and proton leak. (B) Quantitative real-time PCR (qRT-PCR) validation of the partial inhibition of BMP8B by means of si-RNA pools against BMP8B in MiaPaca-2 cells. (C) Effect of marigold SFE on the expression levels of BIP and CHOP in MiaPaca-2 cells transfected with si-RNA pools against BMP8B or si-RNA-scramble. (D) Mitochondrial respiration analysis by flux analysis of the OCR in Panc-1 cells transfected with si-RNA pools against BMP8B or si-RNA-scramble, in non-treated cells and marigold SFE-treated cells (1 × IC 50 , 2 × IC 50 ). (E) qRT-PCR validation of partial inhibition of BMP8B by means of si-RNA pools against BMP8B in Panc-1 cells. (F) Effect of marigold SFE on the expression levels of BIP and CHOP in Panc-1 cells transfected with si-RNA pools against BMP8B or si-RNA-scramble. *Indicates statistical differences referred to control non-treated cell transfected with siRNA scramble: * P < 0.01; ** P < 0.05, *** P < 0.005, **** P < 0.001. # Indicates statistical differences between siRNA BMP8B and siRNA scramble transfected cells for each condition: # P < 0.05, ## P < 0.01, ### P < 0.005, #### P < 0.001.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was performed in the 7900HT Real-Time PCR System (Life Technologies) using the VeriQuest SYBR Green qPCR Master Mix (Affymetrix, Santa Clara, CA, USA), and Taqman probes were used: Hs01629120_s1, Hs01029413_m1, Hs00245183_m1, and Hs99999901_s1 for BMP8B, TFAP2A, ZFP36L1 , and 18S, respectively; or oligos in the case of the epithelial-to-mesenchymal transition (EMT), stemness and endoplasmic reticulum (ER) stress markers ( displays the list and sequences of the primers used).

Techniques: Inhibition, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing