Journal: Animals : an Open Access Journal from MDPI

Article Title: FABP3 Mediates Lipid Droplet Accumulation and Adhesive Capacity in Bovine Endometrial Epithelial Cells via PGE 2 /PTGER4/PPAR Axis

doi: 10.3390/ani15233417

Figure Lengend Snippet: PGE 2 binds to PTGER4 to regulate FABP3 expression in bEECs: ( A ) The difference in PTGER ( PTGER1 , PTGER2 , PTGER3 and PTGER4 ) expression in bEECs after treatment with P 4 and/or PGE 2 . ( B – D ) The representative images and analysis of PTGER2 and PTGER4 expression in bEECs after treatment with P 4 and/or PGE 2 . ( E , F ) The representative images and analysis of PTGER2 expression in bEECs between shN and shPTGER2 groups after treatment with DMSO or P 4 + PGE 2 . ( G , H ) The representative images and analysis of PTGER4 expression in bEECs between shN and shPTGER4 groups after treatment with DMSO or P 4 + PGE 2 . ( I ) The analysis of FABP3 expression in bEECs after treatment with DMSO, PGE 2 or the inhibitor of PTGER4 ( GW627368 ). The data are means ± SEM of three independent experiments. Statistically significant differences are indicated by asterisks: ** p < 0.01, ns p > 0.05. Bars with different letters are significantly different ( p < 0.05).

Article Snippet: Then, PGE 2 (10 −8 –10 −6 M; 14010, Cayman, Ann Arbor, MI, USA) was added to the medium, and the cells were cultured for 24 h. For inhibitor treatment, 5 and 10 μM PTGER2 inhibitor (TG-155; MedChemExpress, Newark, NJ, USA) or 5 and 10 μM PTGER4 inhibitor ( GW627368 ; Selleck, Houston, TX, USA) were added to the medium 1 h prior to the PGE 2 treatment.

Techniques: Expressing

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: Cytotoxicity of amiodarone (AM) and its major metabolite desethylamiodarone (DEA) in HepG2 cells. HepG2 cells were treated with 6.25–100 μM of amiodarone or desethylamiodarone for 6 or 24 h. The cell viability was measured by a CellTiter-Glo assay at the end of treatments. Data were presented as mean±SD of three independent experiments. *P < 0.05 compared with the cell viability of amiodarone at the same concentration.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Glo Assay, Concentration Assay

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: HPLC-QDa mass spectrometry chromatograms for amiodarone (AM) and its metabolite desethylamiodarone (DEA) in an extract from a human microsomal incubation of 12.5 μM amiodarone for 60 min. The analytes were separated using a Waters e2695 HPLC System and monitored with an ACQUITY QDa detector in a positive-electrospray ionization mode. The chromatogram shows selected ion recording (SIR) channels for (a) amiodarone (m/z 646), its internal standard (b) amiodarone-d4 (m/z 650), (c) desethylamiodarone (m/z 618), and its internal standard (d) desethylamiodarone-d4 (m/z 622).

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Mass Spectrometry, Incubation

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: Desethylamiodarone formation in HepG2Cells overexpressing human CYPs. Different human CYPs overexpressed or empty vector-transduced HepG2 cells were treated with 12.5 μM of amiodarone for 6 h. At the end of treatment, cell pellets were harvested and extracted with ice-cold acetonitrile containing 100 ng/ml of deuterated internal standard desethylamiodarone-d4. Desethylamiodarone was quantified with LC–MS as described in Section 2. Data were presented as mean±SD of three independent experiments. *P < 0.05 compared with that of HepG2/empty vector.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: (A) Effects of CYP1A1 or CYP3A4 inhibition on the formation of desethylamiodarone in human microsomal incubation of amiodarone. Human microsomes were incubated with 12.5 μM of amiodarone or coincubated with α-naphthoflavone (50 μM) or ketoconazole (50 μM) in a total volume of 200 μl for 60 min. The incubations were quenched by adding 1 ml of ice-cold acetonitrile containing deuterated internal standards. The amount of amiodarone and desethylamiodarone in extractions was measured with LC–MS as described in Materials and Methods. Data are presented as mean±SD of three independent experiments. *P < 0.05 amiodarone amount compared to that of cells without CYP inhibitor addition; #P < 0.05 desethylamiodarone formation compared to that of cells without CYP inhibitor addition. (B) Effects of CYP inhibition on the formation of desethylamiodarone in HepG2 cells overexpressing CYP1A1 or CYP3A4. HepG2 cells overexpressing CYP1A1 were treated with 12.5 μM of amiodarone (AM) or co-incubated with α-naphthoflavone (10 μM) for 6 h; and HepG2 cells overexpressing CYP3A4 were treated with 12.5 μM of amiodarone or co-incubated with ketoconazole (10 μM) for 6 h. Cell pellets were harvested and extracted with ice-cold acetonitrile containing deuterated internal standards. The extractions were measured by LC–MS for the amounts of amiodarone and desethylamiodarone amounts. The bar graphs show as mean±SD of three independent experiments. *P < 0.05 versus without inhibition.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Inhibition, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Communications Biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: a Scatter graph of gene expression levels in differentiating Hu5/KD3 cells with ( y -axis) or without DAPT treatment ( x -axis). RNA was extracted from the differentiating cells after 4 days DAPT treatment and analyzed by RNA-seq. FPKM: fragments per kilobase of exons per million reads mapped. b Numbers of genes differentially (>2-fold) expressed in differentiating Hu5/KD3 cells with or without DAPT treatment. c Experimental design and qPCR analysis of NOTCH2 , NOTCH3 , PTGER2 , and PTGER4 in Hu5/KD3 cells with or without DAPT treatment. RNA was extracted from the cells at day 0, day 4, and day 7. n = 3 samples/group. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t -test. The effect size of Pearson’s r correlation ( r ) is shown.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Gene Expression, RNA Sequencing, Two Tailed Test

Journal: Communications Biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: a Experimental design. Hu5/KD3 cells were seeded onto collagen type I-coated 60-mm dishes (25,000 cells/cm 2 ) and cultured in 10% FBS/DMEM for 3 days. Then FACS sorting and qPCR analysis were performed. Sorted cells were replated and cultured for a further 3 days, fixed, and stained with MF20 (muscle myosin heavy chain) and DAPI (nuclei). b FACS plot. Sorted NOTCH3-high (N3+) and NOTCH-negative (N3−) fractions are shown by ellipses. c Representative muscle myosin heavy chain staining of differentiating NOTCH3-negative (N3−) and NOTCH3-positive (N3+) Hu5/KD3 cells. Cells were stained with MF20 (MyHC, red) and nuclei (DAPI, blue). Scale bar = 100 μm. Fusion index, percentage of MYOGENIN-positive cells, and cell numbers are shown as dot plots. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t -test. n = 3 samples/group. Three views/sample were analyzed. Both p value and the effect sizes of Pearson’s r correlation ( r ) are shown. d qPCR for NOTCH2 , NOTCH3 , PTGER2 , PTGER4 , HEY1 , and HES1 of NOTCH3-negative (N3−, blue ellipse in b ) and NOTCH3-positive (N3+, red ellipse in b ) cell fractions. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t -test. n = 3 samples/group. Both p -value and the effect sizes of Pearson’s r ( r ) are shown. e Hu5/KD3 cells were transfected with hNICD3-copGFP plasmid or a parental (empty) plasmid, and 3 days later, copGFP-positive cells were collected by FACS and re-seeded on collagen-coated dishes. Two days later, total RNA was extracted from the cells and qPCR for PTGER2(EP2) and PTGER4(EP4) was performed. Unpaired two-tailed Student’s t -test, n = 3 samples/group.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Staining, Two Tailed Test, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: a Experimental design. Hu5/KD3 cells were seeded onto collagen type I-coated 12-well plates (5 × 10 5 cells/well) and cultured in 10% FBS/DMEM medium with prostaglandin E2 (0.5–100 ng/ml), butaprost (EP2 agonist) (0.01–5 µg/ml), or PE2 antagonist, TG6-10-1 (0.01–10 µM) for 3 days. b Expressions of EP2 (green) and MyHC (MF20, red) on differentiating Hu/KD3 cells w/o DAPT treatment. Scale bar, 100 μm. c Histogram of EP2 signal of mononuclear cells treated with (green) or without (black) DAPT. Y -axis indicates the frequency. Data are shown as mean ± S.D. and analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test. More than 600 cells in each group were measured ( n = 3 samples/group). d Representative MyHC staining of differentiating Hu5/KD3 cells treated with PGE2, butaprost, or TG6-10-1 at indicated concentrations. Scale bar, 100 μm. e Fusion index of Hu5/KD3 cells after treatment with PGE2. f Fusion index of Hu5/KD3 cells after treatment with butaprost. g Fusion index of Hu5/KD3 cells after treatment with TG6-10-1.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Staining

Journal: Communications Biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: a Experimental design of overexpression of EP2 in Hu5/KD3 cells. b Western blotting for EP2 (overexpression: OE1-3) and GAPDH (loading control) 3 days after transfection of the control vector (CMV-DsRed) or CMV-EP2-ires DsRed vectors. Relative expression levels of EP2 (after normalization to GAPDH) are shown below. c Representative image of myotubes (MF20, red) formed by Hu5/KD3 cells after transfection of the control vector (CMV-DsRed) or CMV-EP2-ires DsRed vectors. Scale bar, 100 µm. d Fusion index of ( c ). n = 4 samples/group. Dunnett’s multiple comparisons test. e RT-qPCR for EP2 ( PTGER2 ) of nontarget (control) short-hairpin (sh) RNA plasmid-transfected and EP2 shRNA plasmid-transfected Hu5/KD3 cells. Plasmids were introduced into Hu5/KD3 cells by electroporation. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t -test, n = 3 samples/group. f Immunoblot analysis for EP2 and GAPDH in nontarget and shEP2 plasmid-transfected Hu5/KD3 cells. g EP2 signals were normalized to GAPDH. h Representative immunostaining of myotubes formed by nontarget shRNA plasmid or EP2 shRNA plasmid-transfected Hu5/KD3 cells with anti-myosin heavy chain antibody, MF20 (red) and nuclei (DAPI, blue). Scale bar = 200 μm. i Fusion index of Hu5/KD3 cells four days after transfection of shRNA plasmids. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t -test, n = 4 independent experiments. Each dot is an average of 3 views/sample. Both p -value and the effect sizes of Pearson’s r ( r ) are shown. j Full, uncropped images of western blots in b and f are shown in Supplementary Fig. .

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Western Blot, Control, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, shRNA, Electroporation, Two Tailed Test, Immunostaining

Journal: Communications Biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: a Experimental design. After 6 weeks myogenic induction, muscle progenitors were FACS-sorted and treated with EP2 agonists or an antagonist. b Representative images of myotubes formed by hiPSC-derived muscle progenitors treated with PGE2 (100 ng/ml), butaprost (1 µg/ml), TG6-10-1 (TG, 10 µM), and DAPT (10 µM). Cells fixed with 4% PFA were stained with MF20 (muscle myosin heavy chain, red) and nuclei (DAPI, blue). Scale bar, 200 µm. c , d Fusion index (%) ( c ) and cell numbers (/mm 2 ) ( d ). n = 3 samples/group. Three views/sample were analyzed. Data are shown as mean ± S.D. and compared with controls by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Staining

Journal: Communications Biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: In differentiation conditions, the majority of myogenic cells fuse to form multinucleated myotubes. NOTCH signaling upregulates the expression of EP2 and NOTCH3 in a fraction of muscle progenitors. Prostaglandin E2 activates EP2, and promotes self-renewal of muscle progenitors. The interplay between NOTCH3 signal and EP2 signals remains to be clarified. Fusako Sakai-Takemura et al. report that self-renewal of human muscle progenitors is regulated by NOTCH/EP2 signaling. The findings deepen our understanding on self-renewal of muscle stem cells and contribute to the development of treatments for Duchenne muscular dystrophy.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: eLife

Article Title: Increase of circulating IGFBP-4 following genotoxic stress and its implication for senescence

doi: 10.7554/eLife.54523

Figure Lengend Snippet: Panel a - PGE2 can bind the EP2 receptor. This stimulates Gαs, which activates adenylyl cyclase (AC), which produces cyclic AMP (cAMP). This nucleotide induces protein kinase A (PKA) activation. The PGE2 binding to the EP2 receptor determines the release of Gβγ subunits, which stimulate AKT through phosphatidylinositol 3-kinase (PI3K). EP2 can also promote the activation of the RAS-RAF-MEK pathway through the SRC protein. In the cartoon are depicted the drugs and inhibitors of some key factors belonging to the PGE2 signaling pathways. siEP2: siRNA against EP2 mRNA; siGαs: siRNA against Gαs; PP1: SRC specific inhibitor; LY294002 (LY): PI3K inhibitor; bisindolylmaleimide IX (BSD): PKA inhibitor; U0126 (U0): MEK1/2 specific inhibitor. COX2: cyclooxygenase 2; PXB: parecoxib. Panel b – The IGFBP-4 release following EP2 or Gαs silencing. The picture shows the IGFBP-4 levels in the MSC-conditioned medium 48 hr following H 2 O 2 treatment in the presence of either siRNA against EP2 (siEP2) or against Gαs (siGαs). Control siRNAs were indicated as siCTRL. Membrane staining with Ponceau S acid red was used loading control (LC). The graph shows the densitometric analysis of the IGFBP-4 level. The data are expressed in arbitrary units. The statistical difference (p<0.001) between untreated cells (first column) with sample treated with H 2 O 2 (second column) is indicated with *** symbol. Statistical differences (p<0.001) among H 2 O 2 treated cells (second column) with those treated with different siRNAs (third, fourth, fifth column) are indicated with ### symbol. Panel c – The picture shows the IGFBP-4 levels in MSC conditioned medium 48 hr following H 2 O 2 treatment in presence of either Bisindolylmaleimide IX (BSD) (PKA ⊥), or LY294002 (LY) (PI3K ⊥), or PP1 (SRC ⊥) or U0126 (U0) (MEK1/2 ⊥). Membrane staining with Ponceau S acid red was used loading control (LC). The graph shows the densitometric analysis of the IGFBP-4 level. The data are expressed in arbitrary units. The statistical difference (p<0.001) between untreated cells (first column) with sample treated with H 2 O 2 (second column) is indicated with *** symbol. The statistical differences (p<0.001) among H 2 O 2 treated cells (second column) with those treated with different drugs (from third to sixth column) are indicated with ### symbol.

Article Snippet: We obtained validated siRNAs targeting human EP2 (sc-40171) or Gαs (sc-29328) mRNA, as well as control siRNAs (sc-37007) from Santa Cruz Biotechnology (TX, USA).

Techniques: Activation Assay, Binding Assay, Protein-Protein interactions, Control, Membrane, Staining

Journal: eLife

Article Title: Increase of circulating IGFBP-4 following genotoxic stress and its implication for senescence

doi: 10.7554/eLife.54523

Figure Lengend Snippet: Panel a - Quantitative RT-PCR analysis of the EP receptor expression in MSCs. The mRNA levels were normalized to GAPDH mRNA expression, which was selected as an internal control. The data are expressed as arbitrary units with standard deviation. Panels b, c - Quantitative RT-PCR analysis of EP2 and Gαs expression in MSCs treated with siRNA either against EP2 (siEP2) or against Gαs (siGαs). siCTRL indicates cells incubated with control siRNA. The mRNA levels were normalized to GAPDH mRNA expression, which was selected as an internal control. The data are expressed as arbitrary units with standard error ( ± SD, n = 3, **p<0.01, ***p<0.001). Panel d – Test to evaluate PP1, an inhibitor of SRC activity. We incubated cells with 1 μM PP1 for 24 hr and then collected protein lysates for the western blot analysis. The inhibitory activity of PP1 was evaluated by determining the decrease in PI3K phosphorylation. The picture shows global and phosphorylated PI3K in the basal condition and after the PP1 treatment. Panel e - Test to evaluate U0126, an inhibitor of MEK1/2 activity. We incubated cells with 1 μM U0126 for 24 hr and then collected protein lysates for the western blot analysis. The inhibitory activity of U0126 was evaluated by determining the decrease in ERK phosphorylation. The picture shows the global and phosphorylated ERK in the basal condition and after the U0126 treatment. Panel f - Test to evaluate LY294002, an inhibitor of PI3K activity. We incubated cells with 5 μM LY294002 (LY) for 24 hr and then collected protein lysates for the western blot analysis. The inhibitory activity of LY was evaluated by determining the decrease in AKT phosphorylation. The picture shows the global and phosphorylated AKT in the basal condition and after the LY treatment. Panel g - Test to evaluate Bisindolylmaleimide IX (BSD), an inhibitor of PKA activity. We incubated cells with 5 nM BSD for 24 hr and then collected protein lysates for the western blot analysis. The inhibitory activity of BSD was evaluated by determining the decrease in MEK phosphorylation. The picture shows the global and phosphorylated MEK in the basal condition and after the BSD treatment. Panel h – Test to evaluate the specificity of antibodies against human IGF-IR and IGF-IIR. We performed the western blot analysis on protein lysates obtained from human immortal keratinocyte cells (HACAT) and MSCs. The picture shows the western blot films with unique signals corresponding to the expected bands, either for IGF-IR or IGF-IIR.

Article Snippet: We obtained validated siRNAs targeting human EP2 (sc-40171) or Gαs (sc-29328) mRNA, as well as control siRNAs (sc-37007) from Santa Cruz Biotechnology (TX, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, Standard Deviation, Incubation, Activity Assay, Western Blot, Phospho-proteomics