Journal: BMC Cancer

Article Title: IL-1β induces IL-6 production and increases invasiveness and estrogen-independent growth in a TG2-dependent manner in human breast cancer cells

doi: 10.1186/s12885-016-2746-7

Figure Lengend Snippet: IL-1β induced IL-6 production in MCF7_TG2 cells through the IRAK1, NF-kB, JNK, and PI3K signaling pathway. a MCF7_TG2 cells were treated with IL-1β (10 ng/ml) in the presence of IRAK1/4 inhibitor (20 μM), a NF-kB inhibitor (Bay11-7082, 10 μM), a JNK inhibitor (SP600125, 10 μM), an ERK inhibitor (PD98059, 10 μM), a p38 MAPK inhibitor (SB209580, 10 μM), or a PI3K inhibitor (LY294002, 10 μM) for 48 h. IL-6 levels in culture supernatants were measured by ELISA. b MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. Phospho-p65, p65, Ik-Bα, phospho-JNK, and JNK were detected by Western blot. c MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. IRAK1, IRAK2, and TRAF6 were detected by Western blot. d MCF7_Cont and MCF7_TG2 cells were co-transfected with p3kB-Luc and pRL-TK reporter constructs for 24 h then treated with IL-1β (10 ng/nl) for 18 h. All data shown are representative of three independent experiments

Article Snippet: The following signaling inhibitors were added to some cultures 1 h before IL-1β treatment; IRAK1/4 inhibitor (20 μM; Calbiochem), NF-kB inhibitor (Bay-117082, 10 μM; Calbiochem), JNK inhibitor (SP600125, 10 μM; Calbiochem), ERK inhibitor (PD98059, 10 μM; Alomone labs, Jerusalem, Israel), p38 MAPK inhibitor (SB209580, 10 μM; Cell signaling), and PI3K inhibitor (LY294002, 10 μM; Alomone labs).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Construct

Journal: PLoS ONE

Article Title: Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble β-Amyloid 1-40 through Divergent NMDAR-Dependent Signalling Pathways

doi: 10.1371/journal.pone.0006011

Figure Lengend Snippet: Cultured cortical neurons were pre-treated for 45 min with the indicated pharmacological inhibitors before being treated for 1 h with Aβ (1 µM). Inhibitors were present in the medium during Aβ treatment. (A) PI-3K/mTOR pathway is required for Homer1b declustering. Pre-treatment with wortmannin (Wort, 2 µM) abolished the ability of Aβ to disperse Homer1b clusters (101.1±15.3%, cf. Aβ+Wort and Wort alone). Likewise, inhibition of the PI-3K downstream kinases PKB (with API-2, 30 µM) or mTor (with rapamycin, Rapa, 5 µM) prevented Aβ-induced declustering of Homer1b (90.6±8.4%, cf. Aβ+AP2 and API-2 alone; 95±8.2%, cf. Aβ+Rapa and rapamycin alone). Either the blockade of MEK/ERK pathway by pre-treatment with UO126 (10 µM; 70.9±4.8% UO126+Aβ vs UO126 alone) or cdk-5 inhibition by roscovitine (10 µM;63.7±5.8%, cf. Aβ+Rosco and Rosco alone) pre-treatment did affect Aβ-induced Homer declustering. (B) MEK/ERK pathway, but not PI-3K pathway, is required for Aβ-induced dispersal of Shank1. Pre-treatment with the PI-3K inhibitor wortmannin before Aβ treatment did not affect Aβ-induced Shank cluster dispersal (64.7±10.3%, cf. Wort+Aβ and Wort alone; p<0.05). Notably, wortmannin itself decreased Shank1 (but not Homer1b) cluster size (74.3±10.3%, cf. Wort and vehicle; p<0.05). Two structurally unrelated MEK inhibitors, UO126 (10 µM) and PD98059 (25 µM) prevented Aβ-induced declustering of Shank1 (96.8±12.1%, cf. PD98059+Aβ and PD98059 alone; 92.2±15.4%, cf. UO126+Aβ and UO126 alone). Likewise, the blockade of the Erk targeted kinase RSK by pre-treatment with SL0101-1 blocked Aβ effects (100.4±9.8%, p>0.05). Pre-treatment of neurons with the specific cdk-5 inhibitor roscovitine (Rosco, 10 µM) did not interfere with the ability of Aβ to reduce the size of Shank1 (72.6±10.8%, cf. Rosco+Aβ and Aβ alone) clusters. (C,D) PP2B is required for the dispersal of Homer1b, but not Shank1, clusters by Aβ. Pre-treatment with structurally unrelated PP2B inhibitors FK506 (10 µM) or cyclosporin (Cyclo, 100 µM) significantly blocked (p<0.05) Aβ-induced Homer1b declustering (98.8±10.4%, comparing FK506+Aβ vs. Aβ alone; 83.4±5.8%, comparing Aβ+Cyclo and Cyclo alone; and 61±7.9% in vehicle+Aβ-treated cells; see panel C ); however, all PP2B inhibitors were ineffective in preventing Shank1 declustering after Aβ exposure (61.3±4.5%, cf. FK506+Aβ and FK506 alone; 90.8±12.4%, cf. Cyclo+Aβ and Cyclo alone, see panel D ). For each condition, 8–12 neurons from 3–4 replicate experiments were considered (N = 8–12), and at least 50 synapses on each neuron were evaluated (on average, n = 500 puncta/condition).

Article Snippet: Nifedipine, (±)-verapamil, roscovitine, NiCl 2 , cycloheximide, cyclosporin and FK506 were purchased from Sigma Chemicals (Deisenhofen, Germany); NMDA, Bay-K4688, SL0101-1, MK801, UO126, PD98059, API-2, wortmannin, cantharidine were from Tocris (Bristol, UK); and sodium orthovanadate, rapamycin, TDZT, SU6656 and MG132 were from Calbiochem (La Jolla, CA).

Techniques: Cell Culture, Inhibition

Journal: Shock

Article Title: Inhibition of PAR-2 Attenuates Neuroinflammation and Improves Short-Term Neurocognitive Functions Via ERK1/2 Signaling Following Asphyxia-Induced Cardiac Arrest in Rats

doi: 10.1097/shk.0000000000001516

Figure Lengend Snippet: FIG. 5. Inhibition of ERK1/2 abolished the neuroinflammatory effect of PAR-2 activation at 24 h after ACA. Representative Western blot images (A) and quantitative analysis of PAR-2 (B), ERK1/2 (C), p-ERK1/2 (D), IL-6 (E), and TNF- a (F) in the brain revealed increased protein levels at 24 h following ACA except for ERK1/2 compared to the Sham group. Treatment with FSLLRY-NH2 significantly reduced p-ERK1/2 and proinflammatory cytokine levels compared to the ACA þ vehicle group. Further activation of PAR-2 with AC55541 only aggravated the neuroinflammatory response by increasing p-ERK1/2 expression. Potent ERK1/2 inhibitor PD98059 reversed the neuroinflammatory effect of AC55541. Neurologic outcome assessment with NDS at 24 h following ACA (G) revealed that the inhibition of PAR-2 significantly improved neurologic function while AC55541 alone significantly worsened performance compared to the ACA þ vehicle group. This detrimental effect of AC55541 on NDS was reversed by the ERK1/2 inhibitor, PD98059. Data are expressed as mean SD. n ¼ 6/group. ANOVA, Tukey. **P < 0.001 vs. Sham group, *P < 0.05 vs. Sham group, #P < 0.05 vs. ACA þvehicle group, &&P < 0.001 compared to ACA þ AC55541 group, &P < 0.05 compared to ACA þ AC55541 group. ACA indicates asphyxial cardiac arrest.

Article Snippet: Selective PAR-2 activator AC55541 (30 mg/rat) and ERK1/2 inhibitor PD98059 (Santa Cruz Biotechnology, Dallas, Tex; 2 mL of 2 mmol/L) were used for intervention (25).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: Tail Fin Regeneration in Zebrafish: The Role of Non-canonical Crosstalk Between STAT3 and Vitamin D Pathway

doi: 10.7150/ijbs.96400

Figure Lengend Snippet: STAT3 phosphorylations regulate regeneration in vitro and in vivo . A Representative pictures, measurements of scratched area and levels of CXCL1 in supernatants of L929 cells after 6 hours of treatment with vehicle and 1 ng/ml IL-6. B Representative pictures, measurements of scratched area and levels of CXCL1 in supernatants of L929 cells after 24 hours of treatment with vehicle, 5 μM AZD1480 or 12.5 μM PD98059. C Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 0.5 ng/ml IL-6. D Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 200 nM LIF. E Representative pictures and quantification of regenerated area of stat3 +/+ , stat3 +/- and stat3 -/- tail fins cut at 3-dpf and analysed at 3 dpa. F Representative pictures and quantification of regenerated area of wild type zebrafish fins cut at 3-dpf and treated for 3 days either with DMSO, 50 μM AG490 or 12.5 μM PD98059. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Treatments of zebrafish larvae and cells were performed, at the concentration below reported, with the following compounds: LIF (H17002 Sigma); AG490 (T3434 Sigma); PD98059 (Axon 1386, Axon Medchem); vitamin D (D1530 Sigma); AZD1480 (Selleckchem, Houston, TX); IL-6 (R&D Systems).

Techniques: In Vitro, In Vivo