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 Molecular Sciences

Article Title: Novel Osteoblastogenic Activity of Magnolia kobus : The Pharmacological Potential for Osteoporosis

doi: 10.3390/ijms27052472

Figure Lengend Snippet: Involvement of p38 MAPK in ME- and magnolin-induced mineralization in MC3T3-E1 cells. ( A ) ARS staining and ( B ) mineralization assays were performed as described in Materials and Methods. Cells were cultured in DM and treated with ME (100 μg/mL) or magnolin (10 μM) for 21 days in the presence or absence of PD98059 (25 μM) and SB203580 (5 μM). Untreated cells were cultured in 10% FBS-MEM alpha. ( A ) ARS staining images were photographed using a microscope (original magnification ×40). Results are presented as the fold-increase of untreated controls. Statistical significance is indicated ( ### p < 0.001, compared with untreated cells; *** p < 0.001, compared with DM-treated cells).

Article Snippet: The following agents were obtained from commercial sources: anti-DLX5 (sc-398150), anti-cathepsin K (sc-48353), and anti-osterix (sc-393325) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-RUNX2 (12556), anti-NFAT2 (NFATc1, 8032), anti-c-Fos (2250), anti-phospho-ERK (T202/Y204) (9101), anti-phospho-p38 MAPK (T180/Y182) (9215), anti-phospho-JNK (T183/Y185) (9251), anti-ERK (9102), anti-p38 MAPK (9212), anti-JNK (9252), anti-phospho-IKKα/β (2697), anti-IKKβ (8943), anti-phospho-IκBα (2859), anti-IκBα (4814), anti-actin (4970) antibodies, and rabbit and mouse IgG-horseradish peroxidase conjugates (Cell Signaling Technology, Beverly, MA, USA); and PD98059 (S1177) and SB203580 (S1076) (Selleck Chemicals, Houston, TX, USA).

Techniques: Staining, Cell Culture, Microscopy

Journal: iScience

Article Title: Mycobacterial Rv1804c binds to the PEST domain of IκBα and activates macrophage-mediated proinflammatory responses

doi: 10.1016/j.isci.2024.109101

Figure Lengend Snippet:

Article Snippet: PD98059 , MCE , Cat# HY-12028.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Reporter Assay, Transfection, Lysis, Western Blot

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: Oncogene

Article Title: Regulation of IMP3 by EGFR Signaling and Repression by ERβ: Implications for Triple Negative Breast Cancer

doi: 10.1038/onc.2011.620

Figure Lengend Snippet: (A) Immunoblots show the effect of blocking EGFR on IMP3 expression using a specific Ab (2 μg /mL) in MDA-MB-231 (left) and MDA-MB-468 (right) cells. Rabbit IgG was used as control. (B) & (C) MDA-MB-231 and MDA-MB-468 cells were treated with MEK1/2 inhibitors u0126 (10 μM) and PD98059 (50 μM) for different time points as indicated in the figure, and the expression of IMP3, as well as pMAPK, was analyzed by immunoblotting. IMP3 mRNA from cells treated with U0126 for 10 hr was assessed by qPCR. (D) MDA-MB-231 cells were transfected with a luciferase reporter construct containing 2.872 kb IMP3 promoter (wild type) in presence or absence of U0126 and PD98059, and luciferase activity was measured 24 h post-transfection. Data represent the mean of three independent experiments. p value (*) < 0.05.

Article Snippet: The MEK1/2 inhibitors U0126 and PD98059 were purchased from LC Laboratories.

Techniques: Western Blot, Blocking Assay, Expressing, Transfection, Luciferase, Construct, Activity Assay