Journal: Science signaling

Article Title: A plasma membrane–associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells

doi: 10.1126/scisignal.adi7861

Figure Lengend Snippet: (A and B) Relative abundance of Ctnnb1 mRNA and the β-catenin target genes Axin2 and Ccnd1 normalized to 18S RNA in POBs from male (A) or female (B) Prkg2-WT or OB Prkg2-KO mice treated with 17β-estradiol (E2) or 8-CPT-cGMP (cGMP) for 24 h. Hprt served as a control. Mean ΔCt values in vehicle-treated WT cells were assigned a value of one. (C) Immunoblotting for ER-α and AR in whole-cell extracts of male and female WT and KO POBs. β-actin and GAPD are loading controls. Quantification of three independent experiments is shown in fig. S8B. (D) Immunoblotting for total and phosphorylated (p) forms of Src, ERK1/2, and Akt in female Prkg2-WT and Prkg2-KO POBs treated with E2 or 8-CPT-cGMP for 10 min. Quantification of 5 independent experiments is shown in fig. S8D. (E) Immunoblots of the indicated proteins in female WT POBs pre-treated with U1026, LY294002, PP2, or PP3 and treated with E2 for 10 min. Quantification of 2 independent experiments is shown in fig. S8E. (F) Relative abundance of Ctnnb1, Axin2, and Ccnd1 transcripts in female WT POBs pretreated with the indicated drugs and treated with E2 for 24 h. Graphs show means ± S.D. of 3 (A and B) or 4 (F) independent experiments; P values for the indicated comparisons by one-way (F) or two-way ANOVA (A and B). Separate, identical gels loaded with equal amounts of cell lysates were run for analysis of total and phospohorylated forms of Src, ERK, and Akt.

Article Snippet: The Src inhibitor PP2 and its inactive analog PP3, the PI3K inhibitor LY294002, and the MEK inhibitor U1026 were from Calbiochem/EMD.

Techniques: Western Blot

Journal: Science signaling

Article Title: A plasma membrane–associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells

doi: 10.1126/scisignal.adi7861

Figure Lengend Snippet: (A and B) Immunofluorescence staining for total AR (A) or AR phosphorylated on Ser515 (B) in POBs from male Prkg2-WT mice or OB Prkg2-KO mice. Cells were treated for 4 h with vehicle, DHT, or the cGMP analog 8-CPT-cGMP, and some cells were pre-treated with the MEK inhibitor U1026. Graphs show the percentage of cells in which nuclear fluorescence was greater than cytosolic fluorescence; at least 200 cells from 3 independent experiments were analyzed per condition, and the threshold for cytosolic fluorescence intensity was defined in untreated WT cells. Effects of U1026 in WT cells were analyzed separately by one-way ANOVA; comparison between WT and KO cells treated with DHT or cGMP was by two-way ANOVA. (C) WT and KO POBs were treated with DHT for the indicated times, with nuclear and cytosolic fluorescence staining for total AR assessed as in (A). (D) Immunoblotting for and quantification of total AR protein in whole cell lysates of WT and KO POBs treated for 24 h with vehicle, DHT or 8-CPT-cGMP. GAPD is a loading control. (E) AR protein abundance assessed by immunoblotting at the indicated times after addition of cycloheximide at time 0 to male WT POBs; cells were treated with vehicle or DHT for 4 h prior to and during incubation with cycloheximide. The graphs in (C) to (E) show means ± S.D of three independent experiments; p values for the indicated comparisons by two-way ANOVA, with **p<0.01 and ***p<0.001 for the comparison between WT and KO cells (C) or between vehicle and DHT (E) at each timepoint.

Article Snippet: The Src inhibitor PP2 and its inactive analog PP3, the PI3K inhibitor LY294002, and the MEK inhibitor U1026 were from Calbiochem/EMD.

Techniques: Immunofluorescence, Staining, Fluorescence, Comparison, Western Blot, Incubation

Journal: Cells

Article Title: Cyclophilin E (CypE) Functions as a Positive Regulator in Osteoblast Differentiation by Regulating the Transcriptional Activity of Runx2

doi: 10.3390/cells12212549

Figure Lengend Snippet: Kinase inhibitors used in the study.

Article Snippet: Mitogen-activated protein kinase (MEK) inhibitor (U1026) , 662005 , Calbiochem (San Diego, CA, USA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling

doi: 10.1074/jbc.M117.793794

Figure Lengend Snippet: Regulatory signaling of CYGB and αSMA expression in HHSteCs upon FGF2 and supplement solution stimulation. A, effect of transcription inhibitors actinomycin D ( act-D ; 2 μg/ml) and α-amanitin (α AMN ; 2 μg/ml), as well as a translation inhibitor, G418 (2 μg/ml), on CYGB expression under supplement solution ( upper ) and 4 ng/ml FGF2 ( lower ) stimulation in HHSteCs. B, effect of actinomycin D on CYGB mRNA expression in HHSteCs stimulated with FGF2 (4 ng/ml). HHSteCs were incubated with the indicated concentrations of actinomycin D for 72 h. *, p < 0.05 compared with 0-h (one-way ANOVA). C, expressions of CYGB and total and phospho-JNK, ERK, Smad2/3, AKT, and c-JUN in HHSteCs were assayed by Western blot analysis after treatment with medium (−) or FGF2 (4 ng/ml), CTGF (80 ng/ml), HGF (20 ng/ml), PDGF (20 ng/ml), and TGF-β1 (5 ng/ml) at the indicated time points. GAPDH was used as a loading control. D, HHSteCs were treated with supplement solution (1×), FGF2 (4 ng/ml), and TGF-β1 (5 ng/ml) for the indicated lengths of time (1, 4, 8, and 24 h). Total levels of phosphorylated JNK ( pJNK, T183/Y185 ), JNK, phosphorylated ERK ( pERK ), and ERK were assessed using Western blot analysis. E, HHSteCs were treated with an MEK inhibitor, U1026 (20 μ m ), a JNK inhibitor, SP600125 (180 n m ), a p38 inhibitor, SB203580 (64 n m ), and an AKT inhibitor, triciribine (260 n m ) 2 h prior to the addition of supplement solution (1×). Cell lysates were analyzed by Western blotting with antibodies against CYGB. F, HHSteCs were pretreated with a JNK inhibitor and an MEK inhibitor at the indicated doses prior to FGF2 (4 ng/ml) treatment. CYGB protein expression disappeared at a high dose of the JNK inhibitor SP600125 ( left ) but was unaltered by the MEK inhibitor U1026 ( right ) in the presence of FGF2 treatment.

Article Snippet: For the neutralizing assay, anti-FGF2/basic FGF antibodies (2 μg/ml, Millipore, Temecula, CA) were incubated with supplement solution-containing medium for 1 h at 37 °C, and the mixture was added to the cells for 72 h. The effects of the following inhibitors of signaling molecules on CYGB and αSMA expression in HHSteCs were examined at their respective optimal concentrations that were determined from references, and as indicated in F : U1026 (MEK inhibitor) ( , ), triciribine (AKT inhibitor) , SB203580 (p38 inhibitor) , and SP600125 (JNK inhibitor) , all from Wako Pure Chemical Industries, Ltd. Actinomycin D, α-amanitin , and G418 ( ) (Wako Pure Chemical Industries, Ltd.) were used for transcription and translation inhibition assays.

Techniques: Expressing, Incubation, Western Blot