Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Scientific Reports

Article Title: Upregulation of BMSCs Osteogenesis by Positively-Charged Tertiary Amines on Polymeric Implants via Charge/iNOS Signaling Pathway

doi: 10.1038/srep09369

Figure Lengend Snippet: Expressions of osteogenic markers (Runx-2, ALP, OCN, and BSP) of BMSCs cultured in the presence and absence of the NOS inhibitor or siRNA-iNOS transfection in the cell culture medium for 3 days are measured by real time PCR relative to GAPDH expression and normalized to the expressions on Blank without the NOS inhibitor and siRNA. (a) eNOS inhibitor (L-NAME). (b) nNOS inhibitor (Sper). (c) iNOS inhibitor (L-Can). (d) siRNA-iNOS transfection. The bottom left illustrates transfection of siRNA-iNOS with GFP gene into BMSCs and microscopic images of the cells before and after siRNA transfection. (*) and (**) denote two statistical significance (p < 0.05 and p < 0.01), respectively, compared to the sample without NOS inhibitors and siRNA.

Article Snippet: In the siRNA inhibition study, the BMSCs were grown to 60% confluence followed by serum starvation for 12 h. The cells were transfected with siRNA-iNOS(GCAGGACAGCACAGGAAAT) with the GFP gene or scrambled control siRNA oligos (Santa Cruz, USA) at a final concentration of 200 nM according to the manufacturer's instructions.

Techniques: Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Expressing

Journal: Hypertension research : official journal of the Japanese Society of Hypertension

Article Title: Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

doi: 10.1038/hr.2016.54

Figure Lengend Snippet: Figure 1 Silencing HDAC1 and HDAC2 inhibits Ang-II-induced cardiac fibroblast proliferation and migration. (a) Expression of HDAC1 and HDAC2 at basal conditions was analyzed by immunoblotting. CF1, CF2: two independent cultures of primary cardiac fibroblasts. Cell extracts from human epithelial cells (HeLa) and mouse embryonic fibroblasts (NIH3T3) served as positive controls (n = 3). (b) Effect of Ang-II dose on activation of HDAC1 and HDAC2 in quiescent CF as analyzed by a colorimetric assay using nuclear protein extracts (100 μg) immunoprecipitated with HDAC1- or HDAC2-specific antibodies. Deacetylase activity was calculated in pmoles min −1 mg−1. Results are expressed as fold change from controls (n = 6). (c) Silencing HDAC1 or HDAC2 attenuates Ang-II-induced CF proliferation. CF transduced with lentiviral HDAC1 or HDAC2 shRNA (moi 0.5 for 24) were treated with Ang-II (10 −7 M) for 48 h, and proliferation analyzed by CyQUANT assay. (d) Silencing HDAC1 or HDAC2 inhibits Ang-II (10 −7 M for 12 h) -induced CF migration. Migration was analyzed using BioCoat Matrigel invasion chambers and MTT assay. (e) Knockdown of HDAC1 and HDAC2 was confirmed by immunoblotting (n = 3). Akt served as an off-target. (f) Silencing HDAC1 or HDAC2 failed to induce cell death. Forty-eight hours after lentiviral transduction, cell death was analyzed by an ELISA that quantifies mono and oligonucleosomal fragmented DNA in cytoplasmic extracts. (b, c, d, f) *Poat least 0.01 vs. untreated, †Poat least 0.05 vs. Ang-II (n = 6-12).

Article Snippet: Anti-vimentin (#sc-373717) antibodies used in fibroblast characterization, anti-caspase-3 (35 kDa, #sc-136219) antibodies and lentiviral GFP shRNA (#sc-45924-V) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Migration, Expressing, Western Blot, Activation Assay, Colorimetric Assay, Immunoprecipitation, Histone Deacetylase Assay, Activity Assay, Transduction, shRNA, CyQUANT Assay, MTT Assay, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Intra‐epithelial non‐canonical Activin A signaling safeguards prostate progenitor quiescence

doi: 10.15252/embr.202154049

Figure Lengend Snippet: Western blot analysis for Smad4 in mouse prostate organoids upon short‐hairpin RNA mediated knockdown (sh‐Smad4) and in control conditions (sh‐Ctrl). Representative stereoscopic images of control (sh‐Ctrl) and Smad4 knocked down (sh‐Smad4) mouse prostate organoids in normal growth condition (ENRAD) and following A83‐01 withdrawal (‐A83‐01). Scale bar = 1 mm. Source data are available online for this figure.

Article Snippet: The following plasmids were used: pLenti‐AIB‐EGFP (kindly donated by Massimo Pizzato), pSUPER‐retro‐puro‐Smad4 (Addgene #89829), and pSUPER‐retro‐puro‐GFP shRNA (Addgene #30519), MISSION ® shRNA Lentiviral Transduction Particles (AcvrIb, Merck SHCLNV‐TRCN0000022578, SHCLNV‐TRCN0000345027).

Techniques: Western Blot, shRNA, Knockdown, Control