human adipokine antibody array  (R&D Systems)

Journal: Journal of hepatology

Article Title: Hepatic stellate cell activation promotes alcohol-induced steatohepatitis through Igfbp3 and SerpinA12

doi: 10.1016/j.jhep.2020.02.005

Figure Lengend Snippet: (A) Representative BODIPY® staining images and quantification from HepG2Cyp2E1 cells treated with (I) medium, (II) control HSC supernatant, (III) siRNA mediated NRP-1 knockdown HSC supernatant. (III) shows decreased lipid droplet formation in vitro (n=6–9, 63X, one-way ANOVA). (B) Top ten down/upregulated molecules from adipokine and inflammatory protein array from NRP-1 KD HSC supernatant compared to controls. IGFBP3 and SerpinA12 were the most significantly decreased and increased proteins respectively (n=3, p<0.05, unpaired t-test). (C) Igfbp3 mRNA and protein expression in NRP-1 KD HSC is lower than human WT HSC on qPCR and WB (n=6–9, p<0.05, unpaired t-test). (D) SerpinA12 mRNA expression in NRP-1 KD HSC is higher than human WT HSC (n=6–9, p<0.05, unpaired t-test).

Article Snippet: Supernatant and lysate from control or NRP-1 knockdown (KD) human HSC samples were subjected to human cytokines and adipokines antibody-based array (ARY024; R&D Systems, USA) according to manufacturer’s instructions and quantitated using ImageJ-software (NIH).

Techniques: Staining, Control, Knockdown, In Vitro, Protein Array, Expressing

Journal: Science Advances

Article Title: Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes

doi: 10.1126/sciadv.aay6721

Figure Lengend Snippet: ( A ) Oil red O–stained and ( B ) phase-contrast microscopic images of HASCs treated with 1 × 10 9 particles/ml of P-EV, D-EV, and BD-EV for 2 weeks. Differentiation medium (DM) and growth medium were used as positive control and negative controls, respectively. Scale bars, 100 μm. ( C ) Oil red O intensity of lipid droplets in each group. All values are expressed as the means ± SD of four independent trials (** P < 0.0001). ( D ) Adipokines within P-EV versus D-EV and BD-EV measured using adipokine array (* P < 0.05). Both down-regulated and up-regulated adipokines were identified in each EV. ( E ) Fluorescent plots for adipokine array. N.S., not significant; a.u., arbitrary units; ANGPTL4, angiopoietin-like 4.

Article Snippet: We analyzed adipokines in P-EV, D-EV, and BD-EV using a human adipokine antibody array kit (RayBiotech, Norcross, GA, USA) according to the manufacturer’s protocol.

Techniques: Staining, Positive Control

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: Key Resources Table

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-CD44 Cell Signaling Technology Cat# 3570S; clone 156-3C11 Mouse monoclonal anti-LEPR Sigma Aldrich Cat# SAB1402837-100UG; clone 2H5 Rabbit polyclonal anti-CPT1B Abcam Cat# ab104662 Rabbit polyclonal anti-STAT3 Santa Cruz Biotech Cat# SC-482 Rabbit polyclonal anti-pY705-STAT3 Cell Signaling Technology Cat#9145S Rabbit polyclonal anti-GAPDH Santa Cruz Biotech Cat#sc-25778 Rabbit polyclonal anti-phospho-Jak2 (Tyr1007/1008) Cell Signaling Technology Cat# 3771S RNA polymerase Sigma Aldrich Cat#05-952 Normal rabbit IgG Santa Cruz Biotech Cat# SC-2027 Leptin neutralizing antibody: Mouse monoclonal anti-LEPR R&D Systems, Inc. Cat# AF497 Bacterial and Virus Strains N/A Biological Samples Breast cancer patient specimens City of Hope National Cancer Center N/A Chemicals, Peptides, and Recombinant Proteins Recombinant LEPR BD Pharmingen Cat# 564376 Recombinant CCR2 BD Pharmingen Cat# 561744 Recombinant CXCR1 BD Pharmingen Cat# 555939 Recombinant CXCR2 BD Pharmingen Cat# 551126 [ 3 H]-palmitic acid PerkinElmer Inc Cat# NET043001MC [ 3 H]-Acetyl Coenzyme A PerkinElmer Inc Cat# NET290050UC Acetyl-coA Sigma Aldrich Cat# A2056 Myristic acid Sigma Aldrich Cat# M3128 paclitaxel Sigma Aldrich Cat# T7402 Etomoxir Sigma Aldrich Cat# 236020 Accutase Millipore Cat# SCR005 Critical Commercial Assays Imprint Chromatin-Immunoprecipitation kit Sigma Aldrich Cat# CHP1-96RXN Adipokine array R&D Systems Cat# ARY024 ChIPAb+ RNA Pol II - ChIP Validated Antibody and Primer Set Sigma Aldrich Cat#17-620 RNeasy Plus Mini Kit Qiagen Cat#74134 iScript cDNA Synthesis Kit Bio-Rad Cat#1708890 iQ SYBR Green Supermix Bio-Rad Cat#1708880 Deposited Data N/A Experimental Models: Cell Lines MCF10A ATCC CRL-10317 MCF-7 ATCC HTB-22 MDA-MB-468 ATCC HTB-132 MDA-MB-231-Parental This paper N/A MDA-MB-231-Resistant This paper N/A BBM2 Neman and Jandial, 2004 N/A BBM3 Neman and Jandial, 2004 N/A Hs578T ATCC HTB-126 MDA-MB-436 ATCC HTB-130 HCC1500 ATCC CRL-2329 BT20 ATCC HTB-19 Preadipocyte Zen-Bio BR-F Experimental Models: Organisms/Strains Mouse: FVB/N-Tg(MMTV-PyVT)634Mul/J The Jackson Laboratory Cat# 002374 Oligonucleotides Stat3 siRNA (h) Santa Cruz Biotech Cat# sc-29493 Please see supplemental table 1 for primer sequence Recombinant DNA pRC-CMV-STAT3C This paper N/A pGL 3.1 Promega Cat# E1751 pGL3.1-CPT1B This paper N/A Software and Algorithms Oncomine Rhodes and Chinnaiyan, 2004. https://www.oncomine.org/ Open in a separate window Key Resources Table Inhibition of FAO preferentially eliminates BCSCs JAK/STAT3 activates FAO through transcription of CPT1B Adipocyte-derived leptin is critical for JAK/STAT3-FAO in BCSCs Targeting FAO/leptin inhibits BCSCs, chemoresistance and breast tumor growth

Techniques: Recombinant, Chromatin Immunoprecipitation, SYBR Green Assay, Sequencing, Software

Journal: Molecular and Cellular Biology

Article Title: Obesity and p16 INK4A Downregulation Activate Breast Adipocytes and Promote Their Protumorigenicity

doi: 10.1128/MCB.00101-17

Figure Lengend Snippet: Breast adipocytes from obese females are active. (A) Adipocytes (Obadi-1 to -4 and Leadi-1 to -4) were seeded in SFM in the upper wells of the CIM plates, and the migration/invasion abilities were assessed for 24 h. For proliferation, adipocytes were seeded in complete medium in the E-plate, and the proliferation rate was assessed by the xCELLigence RTCA system. (B and C) Cell extracts were prepared from the indicated adipocytes and used for immunoblotting. (D) SFCM from the indicated adipocytes was applied onto human adipokine array membranes. The circles indicate cytokines with higher levels in Obadi than in Leadi adipocytes. (E) SFCM from three Obadi and three Leadi cells was utilized to assess the level of the indicated adipokines by ELISA. Error bars represent means ± SD of values from three Obadi and three Leadi cells from three different experiments. *, P ≤ 0.00021.

Article Snippet: Furthermore, serum-free conditioned medium (SFCM) was collected from three Obadi and three Leadi cells and then applied to Ray Bio human adipokine antibody arrays.

Techniques: Migration, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Obesity and p16 INK4A Downregulation Activate Breast Adipocytes and Promote Their Protumorigenicity

doi: 10.1128/MCB.00101-17

Figure Lengend Snippet: p16 downregulation activates Leadi cells. (A) Mature adipocytes from three Leadi cells were transfected with either an shRNA targeting CDKN2A (Leadi-sh) or the control plasmid (Leadi-C), and cell lysates were prepared and used for immunoblotting. (B) Cells were tested for their proliferation and migration/invasion capacities. The graphs are representative of different experiments. (C) SFCM from the indicated cells was collected after 48 h and applied onto human adipokine antibody array membranes. The circles indicate cytokines with higher levels in Leadi1-sh than in Leadi1-C cells. (D) SFCM was collected from three Leadi-C and three Leadi-sh cells and then utilized to assess the level of the highly secreted adipokines by ELISA. Error bars represent means ± SD of values from three Leadi-C and three Leadi-sh cells from three independent experiments. *, P ≤ 0.0012.

Article Snippet: Furthermore, serum-free conditioned medium (SFCM) was collected from three Obadi and three Leadi cells and then applied to Ray Bio human adipokine antibody arrays.

Techniques: Transfection, shRNA, Plasmid Preparation, Western Blot, Migration, Ab Array, Enzyme-linked Immunosorbent Assay