u0126 Search Results


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  • 99
    Millipore u0126
    BaP-induced vimentin protein expression is modulated by ERK activation. Cells were seeded in 60-mm plates and treated with 4 µM BaP or 20 µM <t>U0126</t> for 48 h. Western blot analysis was performed to analyze the level of protein expression. BaP, benzo(a)pyrene; ERK, extracellular signal-regulated kinase; p, phosphorylated; Akt, protein kinase B.
    U0126, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc u0126
    Effect of <t>U0126</t> on osteoblastic differentiation, APN secretion and angiogenic potential. (A) Mineralized matrix deposition was measured on day 21 by Alizarin red S. (B) The mRNA expression of ALP, RUNX2, and OCN was analyzed on day 14 by RT-PCR. GAPDH was used as the internal control. (C) ALP activity was measured on day 14 using ALP assay kit. (D) APN concentration was measured on day 14 by a human APN ELISA kit. (E) The VEGF level was measured by human VEGF ELISA kit on day 14. (F) The tube formation of HUVECs was analyzed at 24 h. Bar: 300 μm. **P
    U0126, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris u0126
    SCFA-dependent GPR43 signaling confers GI GVHD protection through the ERK–NLRP3 pathway. B6 WT and Gpr43 −/− mice received BMT from syngeneic or allogeneic donors. a Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 normalized to the presence of β-actin in IECs (CD326 + ) from allogeneic recipients 14 days after BMT ( n = 4–5 each, two-tailed Mann–Whitney U test). b Serum IL-18 levels at day 7 and 14 after BMT ( n = 4 WT, n = 5 Gpr43 −/− , two-tailed unpaired t -test). c Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 at day 14 after BMT in IECs (CD326 + ) from allogeneic recipients. Mice were treated with vehicle or propionate (15 mg kg −1 per day) from day 0 ( n = 4 WT, n = 3 Gpr43 −/− , two-tailed Mann–Whitney U test). d Serum IL-18 levels from allogeneic recipients treated with vehicle or propionate (15 mg kg −1 per day) at day 7 and 14 after BMT ( n = 5 WT each, n = 4 Gpr43 −/− each, two-tailed unpaired t -test). e Immunohistochemical staining of IL-18 in colon sections from WT B6 and Gpr43 −/− recipients 14 days after BMT treated with vehicle or propionate (15 mg kg −1 per day) from day 0 ( n = 3–5 each). f IL-22 levels in serum, homogenized tissues from syngeneic and allogeneic recipients at day 14 after BMT ( n = 6 WT syn, n = 4 Gpr43 −/− syn, n = 5 WT allo, n = 10 Gpr43 −/− allo, two-tailed unpaired t -test). g WT B6 and Gpr43 −/− mice received oral administrations of vehicle or propionate (15 mg kg −1 per day) for 3 days. Representative immunoblots and densitometric analysis of phosphorylated ERK normalized to total ERK in IECs (CD326 + ) after treatment ( n = 4 WT each, n = 3 Gpr43 −/− each, two-tailed Mann–Whitney U test). h Colon explants from WT B6 mice were pretreated with <t>U0126</t> (10 μM) for 30 min. Then tissues were primed with LPS (0.1 μg per ml) for 2 h and activated with butyrate or propionate (1 mM) for 2 h. Supernatants were assayed for IL-18 by ELISA ( n = 3 each, two-tailed unpaired t -test). Data are representative of two to three experiments. * P
    U0126, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 773 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories u0126
    Experimental protocol. A total of 48 rats were allocated into 3 surgery groups: sham, IR+vehicle, and <t>IR+U0126.</t> Rats in the vehicleand U0126 -treatment groups were given intraperitoneal injections with 500 μl DMSO or U0126 (30 mg/kg), respectively, 30 min before ischemia and again subcutaneously 6 hours after reperfusion. Rats were euthanized after either 3 h or 24 h of reperfusion.
    U0126, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Beyotime u0126
    Propofol could inhibit Drp1 phosphorylation through decreasing ERK activation in H9c2 cells during OGD/R. (A) Representative bands of pERK, ERK, pDrp1, Drp1 and Mfn1 by western blotting. (B) With the increase of propofol concentration (10, 20 μM), the levels of pERK were significantly decreased during OGD/R injury. (C) <t>U0126</t> can markedly inhibit ERK phosphorylation. (D,F) The levels of pDrp1 (S616) during OGD/R were significantly decreased by 20 μM propofol and U0126. (E) There was no significant difference in the levels of Mfn1 between OGD/R group without propofol and OGD/R group with U0126. The data are presented as the mean ± SD of at least three independent experiments. ∗∗ P
    U0126, supplied by Beyotime, used in various techniques. Bioz Stars score: 95/100, based on 432 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA u0126
    The effect of colony stimulating factor 1 receptor inhibitor, sunitinib, and <t>U0126</t> on Mono-Mac 1 cell cycle distribution Mono-Mac 1 cells were treated for 24 hours with medium alone (A), cFMS-I (B), sunitinib (C), and U0126 (D).
    U0126, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cayman Chemical u0126
    Sensitivity to SFK / MEK inhibition in IM ‐resistant cells. (A) FACS analysis of the percentage of Annexin V‐ FITC ‐positive (apoptotic) cells in a population of K562 and K562‐ STI ‐R cells cultured with 25 μ m IM , 25 μ m <t>U0126,</t> 100 n m dasatinib, the combination of 25 μ m IM and 25 μ m U0126, and the combination of 100 n m dasatinib and 25 μ m U0126. Data are shown as the mean and 1 SD for three independent analyses. * P
    U0126, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 94/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore u0124
    SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific <t>MEK1/2</t> inhibitor <t>U0126</t> abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.
    U0124, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH u0126
    TGF-β1 induces MMP-9 expression and activation in the rat primary cultured astrocytes . (A) Time dependence of TGF-β1-induced MMP-9 expression and activation. Cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. The conditioned media were collected and analyzed MMP-9 activity by gelatin zymography (upper panel). For MMP-9 protein level, conditioned media were immunoprecipitated with an anti-MMP-9 antibody and analyzed by western blot (lower panel). (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), <t>U0126</t> (10 μM), SB202190 (SB20, 10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then treated with TGF-β1 for 24 h. (C) Schematic pathway for TGF-β1-induced ROS-dependent MMP-9 expression and cell migration in RBA-1 cells. Each solid line and arrow represents a step in an activating pathway. TGF-β1 induces MMP-9 expression via TGF-β receptor, ROS-dependent activation of ERK1/2 and JNK1/2, and transcription factor NF-κB pathway, which results in the promotion of cell migration in RBA-1 cells.
    U0126, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    InvivoGen u0126
    Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or <t>U0126</t> (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or pretreated with wortmannin or U0126 at the indicated doses for 1 h at 37°C (G) or transfected with or without siRNAs against PI3K p85α or MEK (H) and then incubated with or without PSaV VLPs. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.
    U0126, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co u0126
    MEK1 maintains liver CSC self-renewal dependent on SIRT1 Non-CSCs (Huh7-Nanog Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM <t>U0126</t> or DMSO for 48 hours. Liver CSCs (Huh7-Nanog Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs ( C ) and non-CSCs ( A ) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs ( D ) and non-CSCs ( B ) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. ( E – F ) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = 0.52 × (W) 2 × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.
    U0126, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    u0126  (Abcam)
    98
    Abcam u0126
    MEK1 maintains liver CSC self-renewal dependent on SIRT1 Non-CSCs (Huh7-Nanog Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM <t>U0126</t> or DMSO for 48 hours. Liver CSCs (Huh7-Nanog Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs ( C ) and non-CSCs ( A ) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs ( D ) and non-CSCs ( B ) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. ( E – F ) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = 0.52 × (W) 2 × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.
    U0126, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals u0126 etoh
    Effects of <t>U0126,</t> oxaliplatin and 5-FU on SW48 cells. Cell viability was measured using CCK-8 assay and is represented as the percentages of the untreated group value. (A) CCK-8 was performed following the treatment of SW48 and NCI-H508 cells with increasing concentrations of U0126 for 72 h. There was a statistical difference between the two groups (*P
    U0126 Etoh, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM u0126
    Effect of 0.01–0.2% sericin ( A ) and 0–20 μM <t>U0126</t> ( B ) on the phosphorylation of ERK1/2 in HCE-T cells. ( A ) 0.01–0.2% sericin was dissolved in 0.05% DMSO, and treated for 24 h. ( B ) 0–20 μM U0126 was dissolved in 0.05% DMSO, and treated for 24 h. The phosphorylation of ERK1/2 was enhanced with the increase in sericin concentration; 20 μM U0126 prevented the phosphorylation of ERK1/2 in HCE-T cells.
    U0126, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Albuterol chemical reference substance
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    N/A
    U0126 is a highly selective inhibitor of MEK1 IC50 of 72nM and MEK2 IC50 of 58nM that antagonizes AP 1 transcriptional activity U0216 helps maintain human pluripotent stem cells in
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    N/A
    Potent and selective non competitive inhibitor of MAP kinase kinase MEK Inhibits MEK 1 and MEK 2 IC₅₀ values of 0 07 and 0 06 µM respectively with little or
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    Image Search Results


    BaP-induced vimentin protein expression is modulated by ERK activation. Cells were seeded in 60-mm plates and treated with 4 µM BaP or 20 µM U0126 for 48 h. Western blot analysis was performed to analyze the level of protein expression. BaP, benzo(a)pyrene; ERK, extracellular signal-regulated kinase; p, phosphorylated; Akt, protein kinase B.

    Journal: Oncology Letters

    Article Title: Benzo(a)pyrene promotes Hep-G2 cell migration and invasion by upregulating phosphorylated extracellular signal-regulated kinase expression

    doi: 10.3892/ol.2018.8379

    Figure Lengend Snippet: BaP-induced vimentin protein expression is modulated by ERK activation. Cells were seeded in 60-mm plates and treated with 4 µM BaP or 20 µM U0126 for 48 h. Western blot analysis was performed to analyze the level of protein expression. BaP, benzo(a)pyrene; ERK, extracellular signal-regulated kinase; p, phosphorylated; Akt, protein kinase B.

    Article Snippet: A total of 2 ml DMEM (GE Healthcare Bio-Sciences) with 4 µM BaP or 20 µM ERK inhibitor, U0126 (U120-1MG; ≥98% HPLC; Sigma-Aldrich; Merck KGaA), was added to each well.

    Techniques: Expressing, Activation Assay, Western Blot

    ERK activation is required for BaP-induced Hep-G2 cell migration and invasion. Wound-healing assay was (A) performed and (B) quantified to evaluate the potential role of p-ERK in BaP-induced Hep-G2 cell migration. Cells were wounded by scratching and cultured in the presence of BaP (4 µM) or U0126 (20 µM) for 48 h. Images were captured at 0 and 48 h. The healing width was calculated as the wound width at 0 h minus the wound width at 48 h and was normalized to the control. Values are expressed as the mean ± standard deviation. Transwell invasion assay was (C) performed and (D) quantified to assess the potential role of p-ERK in BaP-induced Hep-G2 cell invasion. Cells were harvested and seeded into the upper chamber with Matrigel and were cultured in the presence of BaP (4 µM) or U0126 (20 µM) for 24 h. The number of migrated cells was counted and normalized to the control. Values are expressed as mean ± standard deviation. ERK, extracellular signal-regulated kinase; BaP, benzo(a)pyrene; p-ERK, phosphorylated ERK.

    Journal: Oncology Letters

    Article Title: Benzo(a)pyrene promotes Hep-G2 cell migration and invasion by upregulating phosphorylated extracellular signal-regulated kinase expression

    doi: 10.3892/ol.2018.8379

    Figure Lengend Snippet: ERK activation is required for BaP-induced Hep-G2 cell migration and invasion. Wound-healing assay was (A) performed and (B) quantified to evaluate the potential role of p-ERK in BaP-induced Hep-G2 cell migration. Cells were wounded by scratching and cultured in the presence of BaP (4 µM) or U0126 (20 µM) for 48 h. Images were captured at 0 and 48 h. The healing width was calculated as the wound width at 0 h minus the wound width at 48 h and was normalized to the control. Values are expressed as the mean ± standard deviation. Transwell invasion assay was (C) performed and (D) quantified to assess the potential role of p-ERK in BaP-induced Hep-G2 cell invasion. Cells were harvested and seeded into the upper chamber with Matrigel and were cultured in the presence of BaP (4 µM) or U0126 (20 µM) for 24 h. The number of migrated cells was counted and normalized to the control. Values are expressed as mean ± standard deviation. ERK, extracellular signal-regulated kinase; BaP, benzo(a)pyrene; p-ERK, phosphorylated ERK.

    Article Snippet: A total of 2 ml DMEM (GE Healthcare Bio-Sciences) with 4 µM BaP or 20 µM ERK inhibitor, U0126 (U120-1MG; ≥98% HPLC; Sigma-Aldrich; Merck KGaA), was added to each well.

    Techniques: Activation Assay, Migration, Wound Healing Assay, Cell Culture, Standard Deviation, Transwell Invasion Assay

    Effect of U0126 on osteoblastic differentiation, APN secretion and angiogenic potential. (A) Mineralized matrix deposition was measured on day 21 by Alizarin red S. (B) The mRNA expression of ALP, RUNX2, and OCN was analyzed on day 14 by RT-PCR. GAPDH was used as the internal control. (C) ALP activity was measured on day 14 using ALP assay kit. (D) APN concentration was measured on day 14 by a human APN ELISA kit. (E) The VEGF level was measured by human VEGF ELISA kit on day 14. (F) The tube formation of HUVECs was analyzed at 24 h. Bar: 300 μm. **P

    Journal: BMB Reports

    Article Title: Human amnion-derived mesenchymal stem cells induced osteogenesis and angiogenesis in human adipose-derived stem cells via ERK1/2 MAPK signaling pathway

    doi: 10.5483/BMBRep.2018.51.4.005

    Figure Lengend Snippet: Effect of U0126 on osteoblastic differentiation, APN secretion and angiogenic potential. (A) Mineralized matrix deposition was measured on day 21 by Alizarin red S. (B) The mRNA expression of ALP, RUNX2, and OCN was analyzed on day 14 by RT-PCR. GAPDH was used as the internal control. (C) ALP activity was measured on day 14 using ALP assay kit. (D) APN concentration was measured on day 14 by a human APN ELISA kit. (E) The VEGF level was measured by human VEGF ELISA kit on day 14. (F) The tube formation of HUVECs was analyzed at 24 h. Bar: 300 μm. **P

    Article Snippet: The mouse anti-rabbit IgG (L27A9) mAb, phospho-p38 (p-p38) MAPK (Thr180/Tyr182) (D3F9) rabbit mAb, p38 MAPK (D13E1) rabbit mAb, phospho-SAPK/JNK (p-JNK) (Thr183/Tyr185) (81E11) rabbit mAb, SAPK/JNK (JNK) antibody (#9252), phospho-p44/42 (p-ERK1/2) MAPK rabbit mAb, p44/42 MAPK (ERK1/2) rabbit mAb, RUNX2 (D1L7F) rabbit mAb, β-actin (13E5) rabbit mAb, and U0126 (#9903) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, ALP Assay, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Expression of relative proteins in HAMSCs and HASCs cultured with or without U0126 on day 14. (A) Protein expression levels of p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, JNK and RUNX2 were determined by Western blot, β-actin served as an internal control. (B) Protein expression levels of p-ERK1/2, ERK1/2 and RUNX2 were determined by Western blot, β-actin served as an internal control.

    Journal: BMB Reports

    Article Title: Human amnion-derived mesenchymal stem cells induced osteogenesis and angiogenesis in human adipose-derived stem cells via ERK1/2 MAPK signaling pathway

    doi: 10.5483/BMBRep.2018.51.4.005

    Figure Lengend Snippet: Expression of relative proteins in HAMSCs and HASCs cultured with or without U0126 on day 14. (A) Protein expression levels of p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, JNK and RUNX2 were determined by Western blot, β-actin served as an internal control. (B) Protein expression levels of p-ERK1/2, ERK1/2 and RUNX2 were determined by Western blot, β-actin served as an internal control.

    Article Snippet: The mouse anti-rabbit IgG (L27A9) mAb, phospho-p38 (p-p38) MAPK (Thr180/Tyr182) (D3F9) rabbit mAb, p38 MAPK (D13E1) rabbit mAb, phospho-SAPK/JNK (p-JNK) (Thr183/Tyr185) (81E11) rabbit mAb, SAPK/JNK (JNK) antibody (#9252), phospho-p44/42 (p-ERK1/2) MAPK rabbit mAb, p44/42 MAPK (ERK1/2) rabbit mAb, RUNX2 (D1L7F) rabbit mAb, β-actin (13E5) rabbit mAb, and U0126 (#9903) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Cell Culture, Western Blot

    SCFA-dependent GPR43 signaling confers GI GVHD protection through the ERK–NLRP3 pathway. B6 WT and Gpr43 −/− mice received BMT from syngeneic or allogeneic donors. a Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 normalized to the presence of β-actin in IECs (CD326 + ) from allogeneic recipients 14 days after BMT ( n = 4–5 each, two-tailed Mann–Whitney U test). b Serum IL-18 levels at day 7 and 14 after BMT ( n = 4 WT, n = 5 Gpr43 −/− , two-tailed unpaired t -test). c Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 at day 14 after BMT in IECs (CD326 + ) from allogeneic recipients. Mice were treated with vehicle or propionate (15 mg kg −1 per day) from day 0 ( n = 4 WT, n = 3 Gpr43 −/− , two-tailed Mann–Whitney U test). d Serum IL-18 levels from allogeneic recipients treated with vehicle or propionate (15 mg kg −1 per day) at day 7 and 14 after BMT ( n = 5 WT each, n = 4 Gpr43 −/− each, two-tailed unpaired t -test). e Immunohistochemical staining of IL-18 in colon sections from WT B6 and Gpr43 −/− recipients 14 days after BMT treated with vehicle or propionate (15 mg kg −1 per day) from day 0 ( n = 3–5 each). f IL-22 levels in serum, homogenized tissues from syngeneic and allogeneic recipients at day 14 after BMT ( n = 6 WT syn, n = 4 Gpr43 −/− syn, n = 5 WT allo, n = 10 Gpr43 −/− allo, two-tailed unpaired t -test). g WT B6 and Gpr43 −/− mice received oral administrations of vehicle or propionate (15 mg kg −1 per day) for 3 days. Representative immunoblots and densitometric analysis of phosphorylated ERK normalized to total ERK in IECs (CD326 + ) after treatment ( n = 4 WT each, n = 3 Gpr43 −/− each, two-tailed Mann–Whitney U test). h Colon explants from WT B6 mice were pretreated with U0126 (10 μM) for 30 min. Then tissues were primed with LPS (0.1 μg per ml) for 2 h and activated with butyrate or propionate (1 mM) for 2 h. Supernatants were assayed for IL-18 by ELISA ( n = 3 each, two-tailed unpaired t -test). Data are representative of two to three experiments. * P

    Journal: Nature Communications

    Article Title: Microbial metabolite sensor GPR43 controls severity of experimental GVHD

    doi: 10.1038/s41467-018-06048-w

    Figure Lengend Snippet: SCFA-dependent GPR43 signaling confers GI GVHD protection through the ERK–NLRP3 pathway. B6 WT and Gpr43 −/− mice received BMT from syngeneic or allogeneic donors. a Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 normalized to the presence of β-actin in IECs (CD326 + ) from allogeneic recipients 14 days after BMT ( n = 4–5 each, two-tailed Mann–Whitney U test). b Serum IL-18 levels at day 7 and 14 after BMT ( n = 4 WT, n = 5 Gpr43 −/− , two-tailed unpaired t -test). c Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 at day 14 after BMT in IECs (CD326 + ) from allogeneic recipients. Mice were treated with vehicle or propionate (15 mg kg −1 per day) from day 0 ( n = 4 WT, n = 3 Gpr43 −/− , two-tailed Mann–Whitney U test). d Serum IL-18 levels from allogeneic recipients treated with vehicle or propionate (15 mg kg −1 per day) at day 7 and 14 after BMT ( n = 5 WT each, n = 4 Gpr43 −/− each, two-tailed unpaired t -test). e Immunohistochemical staining of IL-18 in colon sections from WT B6 and Gpr43 −/− recipients 14 days after BMT treated with vehicle or propionate (15 mg kg −1 per day) from day 0 ( n = 3–5 each). f IL-22 levels in serum, homogenized tissues from syngeneic and allogeneic recipients at day 14 after BMT ( n = 6 WT syn, n = 4 Gpr43 −/− syn, n = 5 WT allo, n = 10 Gpr43 −/− allo, two-tailed unpaired t -test). g WT B6 and Gpr43 −/− mice received oral administrations of vehicle or propionate (15 mg kg −1 per day) for 3 days. Representative immunoblots and densitometric analysis of phosphorylated ERK normalized to total ERK in IECs (CD326 + ) after treatment ( n = 4 WT each, n = 3 Gpr43 −/− each, two-tailed Mann–Whitney U test). h Colon explants from WT B6 mice were pretreated with U0126 (10 μM) for 30 min. Then tissues were primed with LPS (0.1 μg per ml) for 2 h and activated with butyrate or propionate (1 mM) for 2 h. Supernatants were assayed for IL-18 by ELISA ( n = 3 each, two-tailed unpaired t -test). Data are representative of two to three experiments. * P

    Article Snippet: Tissues were treated with GLPG0974 (GPR43 antagonist, 1 μM, Tocris), sodium propionate and sodium butyrate (0.15, 1, 1.5 mM, Sigma), LPS (0.1 µg per mL, Invivogen), and U0126 (ERK inhibitor, 10 μM, Tocris) as indicated.

    Techniques: Mouse Assay, Western Blot, Two Tailed Test, MANN-WHITNEY, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

    Experimental protocol. A total of 48 rats were allocated into 3 surgery groups: sham, IR+vehicle, and IR+U0126. Rats in the vehicleand U0126 -treatment groups were given intraperitoneal injections with 500 μl DMSO or U0126 (30 mg/kg), respectively, 30 min before ischemia and again subcutaneously 6 hours after reperfusion. Rats were euthanized after either 3 h or 24 h of reperfusion.

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: Experimental protocol. A total of 48 rats were allocated into 3 surgery groups: sham, IR+vehicle, and IR+U0126. Rats in the vehicleand U0126 -treatment groups were given intraperitoneal injections with 500 μl DMSO or U0126 (30 mg/kg), respectively, 30 min before ischemia and again subcutaneously 6 hours after reperfusion. Rats were euthanized after either 3 h or 24 h of reperfusion.

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques:

    Western blot analysis of heart homogenates that contained myocardium and the left anterior descending artery (LAD). A. There was a significant increase in ERK1/2 phosphorylation as normalized to the total ERK1/2 at 3 h of reperfusion, and this was significantly attenuated by treatment with U0126. After 24 h, phospho-ERK levels had returned to baseline and no changes were seen between the groups. Western blot results are shown as dot plots, where each dot represent an individual rat (n = 4–5). Bars and whiskers indicate mean values ± SEM. * P

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: Western blot analysis of heart homogenates that contained myocardium and the left anterior descending artery (LAD). A. There was a significant increase in ERK1/2 phosphorylation as normalized to the total ERK1/2 at 3 h of reperfusion, and this was significantly attenuated by treatment with U0126. After 24 h, phospho-ERK levels had returned to baseline and no changes were seen between the groups. Western blot results are shown as dot plots, where each dot represent an individual rat (n = 4–5). Bars and whiskers indicate mean values ± SEM. * P

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques: Western Blot

    (A) The mRNA expression levels of ET B R in the myocardium and left anterior descending artery (LAD) homogenates in sham, ischemia-reperfusion (IR), and IR+U0126 treated rats at 3 h of IR resulted in up-regulated ET B R mRNA compared to sham. After U0126 treatment, there was no significant upregulation as compared to sham (n = 5–8) (one-way ANOVA with Bonferroni’s multiple comparison test). (B) Western blot analysis revealed an ET B receptor band at approximately 50 kDa. Quantification of ET B receptor immunoreactivity (normalized to GAPDH) revealed increased ET B receptor protein levels ( P

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: (A) The mRNA expression levels of ET B R in the myocardium and left anterior descending artery (LAD) homogenates in sham, ischemia-reperfusion (IR), and IR+U0126 treated rats at 3 h of IR resulted in up-regulated ET B R mRNA compared to sham. After U0126 treatment, there was no significant upregulation as compared to sham (n = 5–8) (one-way ANOVA with Bonferroni’s multiple comparison test). (B) Western blot analysis revealed an ET B receptor band at approximately 50 kDa. Quantification of ET B receptor immunoreactivity (normalized to GAPDH) revealed increased ET B receptor protein levels ( P

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques: Expressing, Western Blot

    (A) mRNA expression levels of ET A R in homogenates of the myocardium and left anterior descending artery (LAD) from sham, IR, or IR and U0126 treated rats at 3 h of reperfusion. No significant changes in ET A R mRNA expression were found between the sham, IR, and IR+U0126 groups (one-way ANOVA post-Bonferroni’s multiple comparison test). Results are expressed as whisker box plots (n = 5–8). (B) Two ET A R-like immunoreactive bands were observed. The upper (higher molecular weight) band, corresponding to full-length ET A R (45 kDa), did not show any changes in overall expression level regardless of treatment and reperfusion duration (one-way ANOVA post-Bonferroni’s multiple comparison test). Results are shown as Whisker boxplots, (n = 4–5). (C) The lower molecular-weight band (30 kDa), generally showed strong up-regulation at 3 h of reperfusion, and this was partly retained at 24 h. U0126 tended to reduce this increase, although this was not significant. Results are shown as Whiske box plots (n = 4–5). (D) A co-immunoprecipitation experiment used 25 μg total protein (3 h IR + vehicle) as input and 5 μg mouse mAb anti-ET-1 (Abcam, ab2786) as the immunoprecipitating antibody. Western blot analysis was performed to detect the ET A R using Sigma pAb rabbit-anti-ET A R #9780 1:500. Specific ET A R-like immunoreactivity was observed for the low molecular weight band (30 kDa).

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: (A) mRNA expression levels of ET A R in homogenates of the myocardium and left anterior descending artery (LAD) from sham, IR, or IR and U0126 treated rats at 3 h of reperfusion. No significant changes in ET A R mRNA expression were found between the sham, IR, and IR+U0126 groups (one-way ANOVA post-Bonferroni’s multiple comparison test). Results are expressed as whisker box plots (n = 5–8). (B) Two ET A R-like immunoreactive bands were observed. The upper (higher molecular weight) band, corresponding to full-length ET A R (45 kDa), did not show any changes in overall expression level regardless of treatment and reperfusion duration (one-way ANOVA post-Bonferroni’s multiple comparison test). Results are shown as Whisker boxplots, (n = 4–5). (C) The lower molecular-weight band (30 kDa), generally showed strong up-regulation at 3 h of reperfusion, and this was partly retained at 24 h. U0126 tended to reduce this increase, although this was not significant. Results are shown as Whiske box plots (n = 4–5). (D) A co-immunoprecipitation experiment used 25 μg total protein (3 h IR + vehicle) as input and 5 μg mouse mAb anti-ET-1 (Abcam, ab2786) as the immunoprecipitating antibody. Western blot analysis was performed to detect the ET A R using Sigma pAb rabbit-anti-ET A R #9780 1:500. Specific ET A R-like immunoreactivity was observed for the low molecular weight band (30 kDa).

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques: Expressing, Whisker Assay, Molecular Weight, Immunoprecipitation, Western Blot

    Serum Troponin T measurements in sham operated, IR+vehicle and IR+U0126 (n = 6 in each group). *** P

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: Serum Troponin T measurements in sham operated, IR+vehicle and IR+U0126 (n = 6 in each group). *** P

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques:

    (A) The mRNA expression levels of ET-1 were sigificantly upregulated at 3 h of IR. U0126 treatment decreased ET-1 mRNA levels significantly compared to vehicle treatment. Results are shown as whisker plots, with whiskers indicating SEM (n = 5–8), ** P ≤ 0.01, *** P ≤ 0.001, one-way ANOVA with Bonferroni’s multiple comparison test. B. Protein immunohistochemistry of LAD (left column, arrows) and myocardium (right column) revealed an increase of ET-1 expression in the vehicle treatment after 24 h of IR. This elavated expression was decreased after treatment with U0126 in both LAD and in the myocardium. (C) Western blot analysis revealed a significant increase in prepro-ET-1 protein levels (30 kDa) at 3 h of IR, and the same trend was observed at 24 h of IR. The results are shown as Whisker box plots, with bars and whiskers indicating mean values ± SEM (n = 4–5). ** P ≤ 0.01, one-way ANOVA with Bonferroni’s multiple comparison test.

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: (A) The mRNA expression levels of ET-1 were sigificantly upregulated at 3 h of IR. U0126 treatment decreased ET-1 mRNA levels significantly compared to vehicle treatment. Results are shown as whisker plots, with whiskers indicating SEM (n = 5–8), ** P ≤ 0.01, *** P ≤ 0.001, one-way ANOVA with Bonferroni’s multiple comparison test. B. Protein immunohistochemistry of LAD (left column, arrows) and myocardium (right column) revealed an increase of ET-1 expression in the vehicle treatment after 24 h of IR. This elavated expression was decreased after treatment with U0126 in both LAD and in the myocardium. (C) Western blot analysis revealed a significant increase in prepro-ET-1 protein levels (30 kDa) at 3 h of IR, and the same trend was observed at 24 h of IR. The results are shown as Whisker box plots, with bars and whiskers indicating mean values ± SEM (n = 4–5). ** P ≤ 0.01, one-way ANOVA with Bonferroni’s multiple comparison test.

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques: Expressing, Whisker Assay, Immunohistochemistry, Western Blot

    Concentration-response curves show the effect of increasing concentrations of Sarafotoxin 6c (S6c) in the left anterior descending artery (LAD) upstream of the ligature, LAD downstream of the ligature, and non-ischemic septal coronary artery (SCA) after treatment with (A) IR and vehicle or (B) U0126. The results are shown as mean values ± SEM (n = 6–7), *** P

    Journal: PLoS ONE

    Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    doi: 10.1371/journal.pone.0174119

    Figure Lengend Snippet: Concentration-response curves show the effect of increasing concentrations of Sarafotoxin 6c (S6c) in the left anterior descending artery (LAD) upstream of the ligature, LAD downstream of the ligature, and non-ischemic septal coronary artery (SCA) after treatment with (A) IR and vehicle or (B) U0126. The results are shown as mean values ± SEM (n = 6–7), *** P

    Article Snippet: Rats were given intraperitoneal injections of U0126 (LC labs, Boston, MA, USA) (30 mg/kg) dissolved in DMSO (Sigma, St Louis, MO, USA) (1.5 ml/kg) or 500 μl DMSO (1.5 ml/kg) 30 min before ischemia and again subcutaneously 6 hours after reperfusion ( ).

    Techniques: Concentration Assay

    Propofol could inhibit Drp1 phosphorylation through decreasing ERK activation in H9c2 cells during OGD/R. (A) Representative bands of pERK, ERK, pDrp1, Drp1 and Mfn1 by western blotting. (B) With the increase of propofol concentration (10, 20 μM), the levels of pERK were significantly decreased during OGD/R injury. (C) U0126 can markedly inhibit ERK phosphorylation. (D,F) The levels of pDrp1 (S616) during OGD/R were significantly decreased by 20 μM propofol and U0126. (E) There was no significant difference in the levels of Mfn1 between OGD/R group without propofol and OGD/R group with U0126. The data are presented as the mean ± SD of at least three independent experiments. ∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Propofol Ameliorates H9c2 Cells Apoptosis Induced by Oxygen Glucose Deprivation and Reperfusion Injury via Inhibiting High Levels of Mitochondrial Fusion and Fission

    doi: 10.3389/fphar.2019.00061

    Figure Lengend Snippet: Propofol could inhibit Drp1 phosphorylation through decreasing ERK activation in H9c2 cells during OGD/R. (A) Representative bands of pERK, ERK, pDrp1, Drp1 and Mfn1 by western blotting. (B) With the increase of propofol concentration (10, 20 μM), the levels of pERK were significantly decreased during OGD/R injury. (C) U0126 can markedly inhibit ERK phosphorylation. (D,F) The levels of pDrp1 (S616) during OGD/R were significantly decreased by 20 μM propofol and U0126. (E) There was no significant difference in the levels of Mfn1 between OGD/R group without propofol and OGD/R group with U0126. The data are presented as the mean ± SD of at least three independent experiments. ∗∗ P

    Article Snippet: U0126 (Beyotime, Haimen, China), MEK1/2 inhibitor, was added to the cells during OGD/R at a concentration of 20 μM.

    Techniques: Activation Assay, Western Blot, Concentration Assay

    The effect of colony stimulating factor 1 receptor inhibitor, sunitinib, and U0126 on Mono-Mac 1 cell cycle distribution Mono-Mac 1 cells were treated for 24 hours with medium alone (A), cFMS-I (B), sunitinib (C), and U0126 (D).

    Journal: Anticancer research

    Article Title: CSF-1R up-regulation is associated with response to pharmacotherapy targeting tyrosine kinase activity in AML cell lines

    doi:

    Figure Lengend Snippet: The effect of colony stimulating factor 1 receptor inhibitor, sunitinib, and U0126 on Mono-Mac 1 cell cycle distribution Mono-Mac 1 cells were treated for 24 hours with medium alone (A), cFMS-I (B), sunitinib (C), and U0126 (D).

    Article Snippet: As shown in , the Mono-Mac 1cells had high ERK activity and CSF-1 induced slight ERK activation. cFMS-I, sunitinib, and U0126 all inhibited ERK activity. sunitinib caused the greatest inhibition of ERK, followed by cFMS-I, and U0126 had the smallest effect on ERK.

    Techniques:

    The effect of colony stimulating factor 1 receptor inhibitor, sunitinib, and U0126 on acute myelogenous leukemia cell line proliferation Mono-Mac 1, THP-1 and U937 cells were treated for 3 days with cFMS-I (A), sunitinib (B), and U0126 (C) * P≤ 0.05 for comparison of treatment at the highest concentration to that of no treatment.

    Journal: Anticancer research

    Article Title: CSF-1R up-regulation is associated with response to pharmacotherapy targeting tyrosine kinase activity in AML cell lines

    doi:

    Figure Lengend Snippet: The effect of colony stimulating factor 1 receptor inhibitor, sunitinib, and U0126 on acute myelogenous leukemia cell line proliferation Mono-Mac 1, THP-1 and U937 cells were treated for 3 days with cFMS-I (A), sunitinib (B), and U0126 (C) * P≤ 0.05 for comparison of treatment at the highest concentration to that of no treatment.

    Article Snippet: As shown in , the Mono-Mac 1cells had high ERK activity and CSF-1 induced slight ERK activation. cFMS-I, sunitinib, and U0126 all inhibited ERK activity. sunitinib caused the greatest inhibition of ERK, followed by cFMS-I, and U0126 had the smallest effect on ERK.

    Techniques: Concentration Assay

    Cell signaling and colony stimulating factor 1 receptor (CSF-1R) receptor function with drug treatment in acute myelogenous leukemia cell lines A: Mono-Mac 1, THP-1 and U937 cells were treated for 24 hours with medium alone or cFMS-I and were re-stimulated with and without colony-stimulating factor 1 (CSF-1) for 20 minutes. Phospho-extracellular-signal regulated kinase (ERK) and total ERK are shown. B: Mono-Mac 1 cells were treated with medium alone, cFMS-I, sunitinib, or U0126 for 24 hours and were re-stimulated with and without CSF-1 for 20 minutes. Phospho-ERK and total ERK are shown. C: Western blot of CSF-1R expression in Mono-Mac 1, THP-1, and U937 cells. D: The expression of CSF-1R in Mono-Mac 1 with and without 5 minutes of CSF-1 treatment cells. E: Mono-Mac 1 cells were pre-treated with cFMS-I or sunitinib for 24 hours and were then treated with CSF-1 for 5 minutes. Immunoprecipitation for CSF-1R followed by western blot or phospho-tyrosine and CSF-1R and F: for total cFMS.

    Journal: Anticancer research

    Article Title: CSF-1R up-regulation is associated with response to pharmacotherapy targeting tyrosine kinase activity in AML cell lines

    doi:

    Figure Lengend Snippet: Cell signaling and colony stimulating factor 1 receptor (CSF-1R) receptor function with drug treatment in acute myelogenous leukemia cell lines A: Mono-Mac 1, THP-1 and U937 cells were treated for 24 hours with medium alone or cFMS-I and were re-stimulated with and without colony-stimulating factor 1 (CSF-1) for 20 minutes. Phospho-extracellular-signal regulated kinase (ERK) and total ERK are shown. B: Mono-Mac 1 cells were treated with medium alone, cFMS-I, sunitinib, or U0126 for 24 hours and were re-stimulated with and without CSF-1 for 20 minutes. Phospho-ERK and total ERK are shown. C: Western blot of CSF-1R expression in Mono-Mac 1, THP-1, and U937 cells. D: The expression of CSF-1R in Mono-Mac 1 with and without 5 minutes of CSF-1 treatment cells. E: Mono-Mac 1 cells were pre-treated with cFMS-I or sunitinib for 24 hours and were then treated with CSF-1 for 5 minutes. Immunoprecipitation for CSF-1R followed by western blot or phospho-tyrosine and CSF-1R and F: for total cFMS.

    Article Snippet: As shown in , the Mono-Mac 1cells had high ERK activity and CSF-1 induced slight ERK activation. cFMS-I, sunitinib, and U0126 all inhibited ERK activity. sunitinib caused the greatest inhibition of ERK, followed by cFMS-I, and U0126 had the smallest effect on ERK.

    Techniques: Western Blot, Expressing, Immunoprecipitation

    Sensitivity to SFK / MEK inhibition in IM ‐resistant cells. (A) FACS analysis of the percentage of Annexin V‐ FITC ‐positive (apoptotic) cells in a population of K562 and K562‐ STI ‐R cells cultured with 25 μ m IM , 25 μ m U0126, 100 n m dasatinib, the combination of 25 μ m IM and 25 μ m U0126, and the combination of 100 n m dasatinib and 25 μ m U0126. Data are shown as the mean and 1 SD for three independent analyses. * P

    Journal: Molecular Oncology

    Article Title: Overexpression of Tpl2 is linked to imatinib resistance and activation of MEK‐ ERK and NF‐κB pathways in a model of chronic myeloid leukemia

    doi: 10.1002/1878-0261.12186

    Figure Lengend Snippet: Sensitivity to SFK / MEK inhibition in IM ‐resistant cells. (A) FACS analysis of the percentage of Annexin V‐ FITC ‐positive (apoptotic) cells in a population of K562 and K562‐ STI ‐R cells cultured with 25 μ m IM , 25 μ m U0126, 100 n m dasatinib, the combination of 25 μ m IM and 25 μ m U0126, and the combination of 100 n m dasatinib and 25 μ m U0126. Data are shown as the mean and 1 SD for three independent analyses. * P

    Article Snippet: For in vitro colony assays, 2 × 103 CD34+ cells were plated in quadruplicate in methylcellulose‐based medium with recombinant cytokines SCF, IL‐3, EPO, GM‐CSF (#H4434; Stem Cell Technologies) in the presence of 5 μm U0126, 50 nm dasatinib, 10 μm PS‐1145, 50 nm dasatinib + 5 μm U0126, 50 nm dasatinib + 10 μm PS‐1145, 50 nm dasatinib + 5 μm U0126 + 10 μm PS‐1145 (Cayman Chemicals, Ann Arbor, MI, USA).

    Techniques: Inhibition, FACS, Cell Culture

    Sensitivity of IM ‐resistant cells to SFK / MEK / IKK inhibition. (A) Western blot analysis was performed on K562 and K562‐ STI ‐R whole‐cell lysates. Protein levels and phosphorylation of NF ‐κB pathway components (Tpl2, NF ‐κB1 (p105, p50), IKK , IkB, and NF ‐κB (p65)) were evaluated. Immunoblots representative of three independent experiments are shown. Histone H3 was used as a loading control. The densitometry results of the western blots along with statistical analyses are summarized in Fig. S6 B. (B) FACS analysis of the percentage of Annexin V‐ FITC ‐positive (apoptotic) cells in a population of K562 and K562‐ STI ‐R cells incubated with 25 μ m U0126, 25 μ m PS ‐1145, 100 n m dasatinib, the combination of 100 n m dasatinib and 25 μ m U0126, the combination of 100 n m dasatinib and 25 μ m PS ‐1145, or the combination of 100 n m dasatinib, 25 μ m U0126, and 25 μ m PS ‐1145. Data are shown as the mean and 1 SD for three independent analyses, * P

    Journal: Molecular Oncology

    Article Title: Overexpression of Tpl2 is linked to imatinib resistance and activation of MEK‐ ERK and NF‐κB pathways in a model of chronic myeloid leukemia

    doi: 10.1002/1878-0261.12186

    Figure Lengend Snippet: Sensitivity of IM ‐resistant cells to SFK / MEK / IKK inhibition. (A) Western blot analysis was performed on K562 and K562‐ STI ‐R whole‐cell lysates. Protein levels and phosphorylation of NF ‐κB pathway components (Tpl2, NF ‐κB1 (p105, p50), IKK , IkB, and NF ‐κB (p65)) were evaluated. Immunoblots representative of three independent experiments are shown. Histone H3 was used as a loading control. The densitometry results of the western blots along with statistical analyses are summarized in Fig. S6 B. (B) FACS analysis of the percentage of Annexin V‐ FITC ‐positive (apoptotic) cells in a population of K562 and K562‐ STI ‐R cells incubated with 25 μ m U0126, 25 μ m PS ‐1145, 100 n m dasatinib, the combination of 100 n m dasatinib and 25 μ m U0126, the combination of 100 n m dasatinib and 25 μ m PS ‐1145, or the combination of 100 n m dasatinib, 25 μ m U0126, and 25 μ m PS ‐1145. Data are shown as the mean and 1 SD for three independent analyses, * P

    Article Snippet: For in vitro colony assays, 2 × 103 CD34+ cells were plated in quadruplicate in methylcellulose‐based medium with recombinant cytokines SCF, IL‐3, EPO, GM‐CSF (#H4434; Stem Cell Technologies) in the presence of 5 μm U0126, 50 nm dasatinib, 10 μm PS‐1145, 50 nm dasatinib + 5 μm U0126, 50 nm dasatinib + 10 μm PS‐1145, 50 nm dasatinib + 5 μm U0126 + 10 μm PS‐1145 (Cayman Chemicals, Ann Arbor, MI, USA).

    Techniques: Inhibition, Western Blot, FACS, Incubation

    Sensitivity of CML CD 34+ cells to SFK / MEK / IKK inhibition. (A) Number of colonies formed in the absence or presence of the combination of dasatinib and U0126, or the combination of dasatinib, U0126, and PS ‐1145. Colonies derived from burst‐forming units erythroid ( BFU ‐E), multilineage granulopoietic, erythroid, macrophage, and megakaryocytic colony‐forming units ( CFU ‐ GEMM ), granulocyte–macrophage colony‐forming units ( CFU ‐ GM ), and macrophage colony‐forming units ( CFU ‐M) were scored in situ after 14 days of incubation of CML CD 34+ cells in methylcellulose‐based medium with cytokines in the presence or absence of the aforementioned combinations of inhibitors. This experiment was performed using CML CD 34+ cells isolated prior to TKI exposure from the bone marrow of three CML patients. Data are shown as the mean and 1 SD . ** P

    Journal: Molecular Oncology

    Article Title: Overexpression of Tpl2 is linked to imatinib resistance and activation of MEK‐ ERK and NF‐κB pathways in a model of chronic myeloid leukemia

    doi: 10.1002/1878-0261.12186

    Figure Lengend Snippet: Sensitivity of CML CD 34+ cells to SFK / MEK / IKK inhibition. (A) Number of colonies formed in the absence or presence of the combination of dasatinib and U0126, or the combination of dasatinib, U0126, and PS ‐1145. Colonies derived from burst‐forming units erythroid ( BFU ‐E), multilineage granulopoietic, erythroid, macrophage, and megakaryocytic colony‐forming units ( CFU ‐ GEMM ), granulocyte–macrophage colony‐forming units ( CFU ‐ GM ), and macrophage colony‐forming units ( CFU ‐M) were scored in situ after 14 days of incubation of CML CD 34+ cells in methylcellulose‐based medium with cytokines in the presence or absence of the aforementioned combinations of inhibitors. This experiment was performed using CML CD 34+ cells isolated prior to TKI exposure from the bone marrow of three CML patients. Data are shown as the mean and 1 SD . ** P

    Article Snippet: For in vitro colony assays, 2 × 103 CD34+ cells were plated in quadruplicate in methylcellulose‐based medium with recombinant cytokines SCF, IL‐3, EPO, GM‐CSF (#H4434; Stem Cell Technologies) in the presence of 5 μm U0126, 50 nm dasatinib, 10 μm PS‐1145, 50 nm dasatinib + 5 μm U0126, 50 nm dasatinib + 10 μm PS‐1145, 50 nm dasatinib + 5 μm U0126 + 10 μm PS‐1145 (Cayman Chemicals, Ann Arbor, MI, USA).

    Techniques: Inhibition, Derivative Assay, In Situ, Incubation, Isolation

    SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific MEK1/2 inhibitor U0126 abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.

    Journal: Oncotarget

    Article Title: Upregulation of SIRT6 predicts poor prognosis and promotes metastasis of non-small cell lung cancer via the ERK1/2/MMP9 pathway

    doi: 10.18632/oncotarget.9750

    Figure Lengend Snippet: SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific MEK1/2 inhibitor U0126 abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.

    Article Snippet: To test the effect of the specific MEK1/2 inhibitor U0126 (Sigma) on the migratory and invasive abilities of cells, A549 and L78 cells were pretreated with 10 μM U0126 for 30 min before migration and invasion assays were performed.

    Techniques: Migration, Activity Assay, Plasmid Preparation, Western Blot, Over Expression, Expressing, Wound Healing Assay, Transwell Assay

    U0126 reversed acid-induced mechanical allodynia in the sham but not OVX rats. ( A ) Intrathecal (i.t.) injection of the ERK inhibitor U0126 (10 μg in 20 μL 5% DMSO) in the ovary-intact (Sham) rats on day 5 after the 2nd AI (PD5) attenuated mechanical allodynia at 3 h and the next day (PD6). This effect was not detected in vehicle-treated rats (Sham-DMSO). ( B ) No difference was observed in OVX rats with i.t. U0126 or vehicle injection. BL, before 1st injection; PD1, day 1 post 2nd AI; PD5-3 h, 3 h post U0126/or vehicle injection; PD6, day 6 post 2nd AI. ## p

    Journal: Scientific Reports

    Article Title: Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain

    doi: 10.1038/s41598-019-39472-z

    Figure Lengend Snippet: U0126 reversed acid-induced mechanical allodynia in the sham but not OVX rats. ( A ) Intrathecal (i.t.) injection of the ERK inhibitor U0126 (10 μg in 20 μL 5% DMSO) in the ovary-intact (Sham) rats on day 5 after the 2nd AI (PD5) attenuated mechanical allodynia at 3 h and the next day (PD6). This effect was not detected in vehicle-treated rats (Sham-DMSO). ( B ) No difference was observed in OVX rats with i.t. U0126 or vehicle injection. BL, before 1st injection; PD1, day 1 post 2nd AI; PD5-3 h, 3 h post U0126/or vehicle injection; PD6, day 6 post 2nd AI. ## p

    Article Snippet: Intrathecal injection of the ERK MAPK inhibitor in female rats To investigate the involvement of ERK activation in nociceptive sensitization, i.t. injection of the ERK inhibitor U0126 (Sigma-Aldrich, Saint Louis, MO) was performed in female rats.

    Techniques: Injection

    Immunofluorescence analysis indicates the effect of ERK inhibitor on acid injection-induced ERK activation in the spinal dorsal horn. U0126 or vehicle (doses as Fig. 7 ) was injected 5 days after the 2nd AI. ( A ) Immunofluorescence reveals the p-ERK distribution in the L4/5 ipsilateral spinal cords before the 1st AI (Pre) and 3 h post U0126 or DMSO injection. Upper panels: Sham; lower panels: OVX. Scale bar: 100 μm. ( B ) Comparisons of p-ERK-ir cell numbers among the sham and OVX groups with DMSO or U0126. Data were analysed on the basis of laminae, spinal segment, and AI side. One-way ANOVA with post hoc Tukey’s test, # p

    Journal: Scientific Reports

    Article Title: Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain

    doi: 10.1038/s41598-019-39472-z

    Figure Lengend Snippet: Immunofluorescence analysis indicates the effect of ERK inhibitor on acid injection-induced ERK activation in the spinal dorsal horn. U0126 or vehicle (doses as Fig. 7 ) was injected 5 days after the 2nd AI. ( A ) Immunofluorescence reveals the p-ERK distribution in the L4/5 ipsilateral spinal cords before the 1st AI (Pre) and 3 h post U0126 or DMSO injection. Upper panels: Sham; lower panels: OVX. Scale bar: 100 μm. ( B ) Comparisons of p-ERK-ir cell numbers among the sham and OVX groups with DMSO or U0126. Data were analysed on the basis of laminae, spinal segment, and AI side. One-way ANOVA with post hoc Tukey’s test, # p

    Article Snippet: Intrathecal injection of the ERK MAPK inhibitor in female rats To investigate the involvement of ERK activation in nociceptive sensitization, i.t. injection of the ERK inhibitor U0126 (Sigma-Aldrich, Saint Louis, MO) was performed in female rats.

    Techniques: Immunofluorescence, Injection, Activation Assay

    DAZAP1 regulates cell proliferation (a) Stable over-expression of DAZAP1 in non-small cell lung carcinoma NCI-H157 cells as detected by western blots. Control cells are stably transfected with vector only. (b) The expression of DAZAP1 inhibited cell proliferation as judged by colony formation assay with H157 cells. A representative picture from the triplicate experiments, the means and s.d. were plotted. (c) DAZAP1 inhibited the anchorage independent cell growth of H157 as judged by soft agar assays. A representative picture from the triplicates and the quantification were shown. The box plot was drawn using 25 and 75 quartiles in Sigma Plot with whiskers indicating max and min value and dots indicating outliers. P values were determined using paired t-test, *=p ≤ 5.0×10 −5 . (d) DAZAP1 inhibited cell migration as judged by wound healing assay. The picture of a representative plate (left panel) and the quantification of percent of gap closure were shown (right panel). Data from three independent assays were performed error bar indicate s.d. (e) The quantification of cell cycle stages (G1, S and G2/M phases) in DAZAP1 over-expressed or control cells. The cell cycle stages were analyzed by flow-cytometry measurement of DNA content. Data from two independent flow analysis, error bar indicate s.d. (f) Expression of endogenous DAZAP1 protein during cell cycle as detected by western blots. Tubulin and canonical cell cycle markers (cyclin A1 and B1) were also probed as controls. (g) An integrated model for DAZAP1 function and the regulation of its activity through the MEK/Erk pathway.

    Journal: Nature communications

    Article Title: The splicing activator DAZAP1 integrates splicing control into MEK/Erk regulated cell proliferation and migration

    doi: 10.1038/ncomms4078

    Figure Lengend Snippet: DAZAP1 regulates cell proliferation (a) Stable over-expression of DAZAP1 in non-small cell lung carcinoma NCI-H157 cells as detected by western blots. Control cells are stably transfected with vector only. (b) The expression of DAZAP1 inhibited cell proliferation as judged by colony formation assay with H157 cells. A representative picture from the triplicate experiments, the means and s.d. were plotted. (c) DAZAP1 inhibited the anchorage independent cell growth of H157 as judged by soft agar assays. A representative picture from the triplicates and the quantification were shown. The box plot was drawn using 25 and 75 quartiles in Sigma Plot with whiskers indicating max and min value and dots indicating outliers. P values were determined using paired t-test, *=p ≤ 5.0×10 −5 . (d) DAZAP1 inhibited cell migration as judged by wound healing assay. The picture of a representative plate (left panel) and the quantification of percent of gap closure were shown (right panel). Data from three independent assays were performed error bar indicate s.d. (e) The quantification of cell cycle stages (G1, S and G2/M phases) in DAZAP1 over-expressed or control cells. The cell cycle stages were analyzed by flow-cytometry measurement of DNA content. Data from two independent flow analysis, error bar indicate s.d. (f) Expression of endogenous DAZAP1 protein during cell cycle as detected by western blots. Tubulin and canonical cell cycle markers (cyclin A1 and B1) were also probed as controls. (g) An integrated model for DAZAP1 function and the regulation of its activity through the MEK/Erk pathway.

    Article Snippet: Addition of MEK inhibitor (U0126, Sigma) was followed 2 h post transfection and cells were collected after 16–18h-post treatment for protein and splicing analysis.

    Techniques: Over Expression, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Colony Assay, Migration, Wound Healing Assay, Flow Cytometry, Cytometry, Activity Assay

    DAZAP1 is regulated by Erk1/2 catalyzed threonine phosphorylation in the CTD ( a ). A series of truncated CTDs of DAZAP1 were tethered to the exon of a splicing reporter to test which regions are critical for its activity. The full length DAZAP1 was used as positive control, and the two Erk1/2 phosphorylation sites (Thr269 and Thr315) are marked by red triangle. The experimental conditions were identical to Fig. 3b . The mean PSI values and s.d. are shown below a representative gel. P values were determined with paired t-test by comparing to vector-only control. In all panels, #: p ≤0.05; *= p ≤0.01; **= p ≤0.002 ; *** = p ≤0.0008; ns= not significant. ( b ) A schematic model showing that DAZAP1 activity is mediated by MEK/Erk pathway (left panel). The tethering experiments similar to panel a were conducted in cells treated with MEK/Erk activator (PMA) or inhibitor (U0126). ( c ). Phosphorylation of DAZAP1 was inhibited by two specific MEK inhibitors as judged by western blot with Phos-tag labeling. The phosphorylated forms of DAZAP1 were indicated with arrows. (d) Mutation of the two DAZAP1 threonine sites phosphorylated by Erk1/2 abolished DAZAP1 activity as judged by tethering experiments. The experiments were conducted in triplicates and the mean and s.d. of PSI values are plotted below a representative gel. The western blot was shown on the right to confirm equal expression between wild type and mutated DAZAP1. The band indicated by the asterisk is likely a degradation product of MS2-DAZAP1. (e) Inhibition of MEK/Erk by U0126 affected the splicing of endogenous DAZAP1 targets FIP1L1 and MELK. A representative gel from duplicated experiments and the relative changes of PSI were plotted with ranges as error bars. ( f ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of DAZAP1. The cells were transfected with tagged DAZAP1 and hnRNP A1 and detected by corresponding antibodies. Bar, 5 μm. The percents of cells with DAZAP1 localized in nucleus (N), cytoplasm (C) or both compartments (N+C) were counted and plotted in the right panel (n=80 for each sample in two independent experiments). ( g ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of endogenous DAZAP1. Scale bar, 2.5 μm.

    Journal: Nature communications

    Article Title: The splicing activator DAZAP1 integrates splicing control into MEK/Erk regulated cell proliferation and migration

    doi: 10.1038/ncomms4078

    Figure Lengend Snippet: DAZAP1 is regulated by Erk1/2 catalyzed threonine phosphorylation in the CTD ( a ). A series of truncated CTDs of DAZAP1 were tethered to the exon of a splicing reporter to test which regions are critical for its activity. The full length DAZAP1 was used as positive control, and the two Erk1/2 phosphorylation sites (Thr269 and Thr315) are marked by red triangle. The experimental conditions were identical to Fig. 3b . The mean PSI values and s.d. are shown below a representative gel. P values were determined with paired t-test by comparing to vector-only control. In all panels, #: p ≤0.05; *= p ≤0.01; **= p ≤0.002 ; *** = p ≤0.0008; ns= not significant. ( b ) A schematic model showing that DAZAP1 activity is mediated by MEK/Erk pathway (left panel). The tethering experiments similar to panel a were conducted in cells treated with MEK/Erk activator (PMA) or inhibitor (U0126). ( c ). Phosphorylation of DAZAP1 was inhibited by two specific MEK inhibitors as judged by western blot with Phos-tag labeling. The phosphorylated forms of DAZAP1 were indicated with arrows. (d) Mutation of the two DAZAP1 threonine sites phosphorylated by Erk1/2 abolished DAZAP1 activity as judged by tethering experiments. The experiments were conducted in triplicates and the mean and s.d. of PSI values are plotted below a representative gel. The western blot was shown on the right to confirm equal expression between wild type and mutated DAZAP1. The band indicated by the asterisk is likely a degradation product of MS2-DAZAP1. (e) Inhibition of MEK/Erk by U0126 affected the splicing of endogenous DAZAP1 targets FIP1L1 and MELK. A representative gel from duplicated experiments and the relative changes of PSI were plotted with ranges as error bars. ( f ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of DAZAP1. The cells were transfected with tagged DAZAP1 and hnRNP A1 and detected by corresponding antibodies. Bar, 5 μm. The percents of cells with DAZAP1 localized in nucleus (N), cytoplasm (C) or both compartments (N+C) were counted and plotted in the right panel (n=80 for each sample in two independent experiments). ( g ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of endogenous DAZAP1. Scale bar, 2.5 μm.

    Article Snippet: Addition of MEK inhibitor (U0126, Sigma) was followed 2 h post transfection and cells were collected after 16–18h-post treatment for protein and splicing analysis.

    Techniques: Activity Assay, Positive Control, Plasmid Preparation, Western Blot, Labeling, Mutagenesis, Expressing, Inhibition, Translocation Assay, Transfection

    TGF-β1 induces MMP-9 expression and activation in the rat primary cultured astrocytes . (A) Time dependence of TGF-β1-induced MMP-9 expression and activation. Cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. The conditioned media were collected and analyzed MMP-9 activity by gelatin zymography (upper panel). For MMP-9 protein level, conditioned media were immunoprecipitated with an anti-MMP-9 antibody and analyzed by western blot (lower panel). (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SB202190 (SB20, 10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then treated with TGF-β1 for 24 h. (C) Schematic pathway for TGF-β1-induced ROS-dependent MMP-9 expression and cell migration in RBA-1 cells. Each solid line and arrow represents a step in an activating pathway. TGF-β1 induces MMP-9 expression via TGF-β receptor, ROS-dependent activation of ERK1/2 and JNK1/2, and transcription factor NF-κB pathway, which results in the promotion of cell migration in RBA-1 cells.

    Journal: Journal of Neuroinflammation

    Article Title: Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways

    doi: 10.1186/1742-2094-7-88

    Figure Lengend Snippet: TGF-β1 induces MMP-9 expression and activation in the rat primary cultured astrocytes . (A) Time dependence of TGF-β1-induced MMP-9 expression and activation. Cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. The conditioned media were collected and analyzed MMP-9 activity by gelatin zymography (upper panel). For MMP-9 protein level, conditioned media were immunoprecipitated with an anti-MMP-9 antibody and analyzed by western blot (lower panel). (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SB202190 (SB20, 10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then treated with TGF-β1 for 24 h. (C) Schematic pathway for TGF-β1-induced ROS-dependent MMP-9 expression and cell migration in RBA-1 cells. Each solid line and arrow represents a step in an activating pathway. TGF-β1 induces MMP-9 expression via TGF-β receptor, ROS-dependent activation of ERK1/2 and JNK1/2, and transcription factor NF-κB pathway, which results in the promotion of cell migration in RBA-1 cells.

    Article Snippet: Actinomycin D, cycloheximide, SB431542, U0126, SB202190, SP600125, helenalin, and Bay11-7082 were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Activation Assay, Cell Culture, Activity Assay, Zymography, Immunoprecipitation, Western Blot, Migration

    The ROS/MAPKs-dependent NF-κB cascade is required for TGF-β1-induced MMP-9 promoter activity . (A) Schematic representation of a 5'-promoter regions of the rat MMP-9 gene fused to the pGL-luciferase reporter gene (pGL-MMP-9-Luc). The translational start site (+1) of the luciferase reporter gene is indicated by an arrow. RBA-1 cells were transiently cotransfected with pGL-MMP9-Luc and pGal encoding for b-galactosidase. After transfection, cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then incubated with TGF-β1 for 16 h. (C) Activation of wild-type (WT) and NF-κB-point-mutated (mt-κB) MMP-9 promoter constructs by TGF-β1. Schematic representation of the different MMP-9-luciferase constructs, either wild-type (WT) or modified by single-point mutation of the NF-κB binding site (upper panel). After overnight cotransfection and incubation with TGF-β1 for 16 h, promoter activities of different MMP-9-promoter constructs were measured as relative MMP-9 promoter activity to b-galactosidase. The relative increase in MMP-9 promoter activity induced by TGF-β1 normalized to that of un-stimulated cells is indicated as fold increase. Data are expressed as mean ± SEM of at least three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways

    doi: 10.1186/1742-2094-7-88

    Figure Lengend Snippet: The ROS/MAPKs-dependent NF-κB cascade is required for TGF-β1-induced MMP-9 promoter activity . (A) Schematic representation of a 5'-promoter regions of the rat MMP-9 gene fused to the pGL-luciferase reporter gene (pGL-MMP-9-Luc). The translational start site (+1) of the luciferase reporter gene is indicated by an arrow. RBA-1 cells were transiently cotransfected with pGL-MMP9-Luc and pGal encoding for b-galactosidase. After transfection, cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then incubated with TGF-β1 for 16 h. (C) Activation of wild-type (WT) and NF-κB-point-mutated (mt-κB) MMP-9 promoter constructs by TGF-β1. Schematic representation of the different MMP-9-luciferase constructs, either wild-type (WT) or modified by single-point mutation of the NF-κB binding site (upper panel). After overnight cotransfection and incubation with TGF-β1 for 16 h, promoter activities of different MMP-9-promoter constructs were measured as relative MMP-9 promoter activity to b-galactosidase. The relative increase in MMP-9 promoter activity induced by TGF-β1 normalized to that of un-stimulated cells is indicated as fold increase. Data are expressed as mean ± SEM of at least three independent experiments (n = 3). * P

    Article Snippet: Actinomycin D, cycloheximide, SB431542, U0126, SB202190, SP600125, helenalin, and Bay11-7082 were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Activity Assay, Luciferase, Transfection, Incubation, Activation Assay, Construct, Modification, Mutagenesis, Binding Assay, Cotransfection

    Involvement of ERK1/2 in TGF-β1-induced MMP-9 expression in RBA-1 cells . (A) Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of U0126. (B) Cells were pretreated with U0126 (10 mM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (C) Time dependence of TGF-β1-stimulated ERK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated time intevals. (D) Cells were treated with TGF-β1 (15 ng/ml) for 10 min in the absence or presence of U0126 (10 μM). The whole cell lysates (C, D) were subjected to 10% SDS-PAGE and analyzed using an anti-phospho-ERK1/2 or anti-GAPDH (as an internal control) antibody. (E) Cells were transfected with an empty vector (pcDNA3, as a control), a dominant negative mutant of ERK1 (ΔERK1) or ERK2 (ΔERK2) for 24 h, and then exposed to TGF-β1 for 16 h. The conditioned media (A, E) were analyzed gelatin zymorgraphy. Data are expressed as mean ± SEM (C) or mean (A, B, D, E) of at least three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways

    doi: 10.1186/1742-2094-7-88

    Figure Lengend Snippet: Involvement of ERK1/2 in TGF-β1-induced MMP-9 expression in RBA-1 cells . (A) Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of U0126. (B) Cells were pretreated with U0126 (10 mM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (C) Time dependence of TGF-β1-stimulated ERK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated time intevals. (D) Cells were treated with TGF-β1 (15 ng/ml) for 10 min in the absence or presence of U0126 (10 μM). The whole cell lysates (C, D) were subjected to 10% SDS-PAGE and analyzed using an anti-phospho-ERK1/2 or anti-GAPDH (as an internal control) antibody. (E) Cells were transfected with an empty vector (pcDNA3, as a control), a dominant negative mutant of ERK1 (ΔERK1) or ERK2 (ΔERK2) for 24 h, and then exposed to TGF-β1 for 16 h. The conditioned media (A, E) were analyzed gelatin zymorgraphy. Data are expressed as mean ± SEM (C) or mean (A, B, D, E) of at least three independent experiments (n = 3). * P

    Article Snippet: Actinomycin D, cycloheximide, SB431542, U0126, SB202190, SP600125, helenalin, and Bay11-7082 were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, SDS Page, Transfection, Plasmid Preparation, Dominant Negative Mutation

    TGF-β1-induced MMP-9 expression is mediated through JNK1/2, but not p38 MAPK . Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of (A) SB202190 or (B) SP600125. (C) Cells were pretreated with SB202190 (SB, 10 μM) or SP600125 (SP, 10 μM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (D) Time dependence of TGF-β1-stimulated JNK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated times. Moreover, cells were treated with TGF-β1 for 4 h in the presence of SP600125 (SP/4, 10 μM). (E) Cells were transfected with an empty vector (pcDNA3, as a control) or dominant negative mutant of p38 MAPK (Δp38) or JNK (ΔJNK) for 24 h, and then exposed to TGF-β1 for 16 h. (F) For cell migration, cells were pretreated with U0126 (10 μM) or SP600125 (10 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. The image is representative of three similar experiments (n = 3). The conditioned media (A, B, E) were analyzed gelatin zymorgraphy, and the whole cell lysates (D) were analyzed using an anti-phospho-JNK1/2 or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM (D) or mean (A, B, C, E) of at least three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways

    doi: 10.1186/1742-2094-7-88

    Figure Lengend Snippet: TGF-β1-induced MMP-9 expression is mediated through JNK1/2, but not p38 MAPK . Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of (A) SB202190 or (B) SP600125. (C) Cells were pretreated with SB202190 (SB, 10 μM) or SP600125 (SP, 10 μM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (D) Time dependence of TGF-β1-stimulated JNK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated times. Moreover, cells were treated with TGF-β1 for 4 h in the presence of SP600125 (SP/4, 10 μM). (E) Cells were transfected with an empty vector (pcDNA3, as a control) or dominant negative mutant of p38 MAPK (Δp38) or JNK (ΔJNK) for 24 h, and then exposed to TGF-β1 for 16 h. (F) For cell migration, cells were pretreated with U0126 (10 μM) or SP600125 (10 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. The image is representative of three similar experiments (n = 3). The conditioned media (A, B, E) were analyzed gelatin zymorgraphy, and the whole cell lysates (D) were analyzed using an anti-phospho-JNK1/2 or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM (D) or mean (A, B, C, E) of at least three independent experiments (n = 3). * P

    Article Snippet: Actinomycin D, cycloheximide, SB431542, U0126, SB202190, SP600125, helenalin, and Bay11-7082 were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Transfection, Plasmid Preparation, Dominant Negative Mutation, Migration

    NF-κB is involved in TGF-β1-induced MMP-9 expression and cell migration in RBA-1 cells . (A) Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of helenalin or Bay11-7082. (B) Cells were pretreated with helenalin (HLN, 1 μM) or Bay11-7082 (Bay, 1 μM) before exposure to TGF-β1 for 6 h. The conditioned media and total RNA were collected and analyzed by gelatin zymography (A) and RT-PCR (B). (C) Time dependence of TGF-β1-stimulated NF-κB p65 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated time intervals. (D) Cells were pretreated with U0126 (U0, 10 μM), SP600125 (SP, 10 μM), NAC (100 μM), or Bay11-7082 (Bay, 1 μM) for 1 h before exposure to TGF-β1 for 1 h. Whole cell lysates were analyzed by western blotting using an anti-phospho-NF-κB-p65 antibody. (E) For cell migration, cells were pretreated with Bay11-7082 (1 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. Representative phase contrast images are shown for 48 h (n = 3). Data are expressed as mean ± SEM (C) or mean (A, B, D) of three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways

    doi: 10.1186/1742-2094-7-88

    Figure Lengend Snippet: NF-κB is involved in TGF-β1-induced MMP-9 expression and cell migration in RBA-1 cells . (A) Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of helenalin or Bay11-7082. (B) Cells were pretreated with helenalin (HLN, 1 μM) or Bay11-7082 (Bay, 1 μM) before exposure to TGF-β1 for 6 h. The conditioned media and total RNA were collected and analyzed by gelatin zymography (A) and RT-PCR (B). (C) Time dependence of TGF-β1-stimulated NF-κB p65 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated time intervals. (D) Cells were pretreated with U0126 (U0, 10 μM), SP600125 (SP, 10 μM), NAC (100 μM), or Bay11-7082 (Bay, 1 μM) for 1 h before exposure to TGF-β1 for 1 h. Whole cell lysates were analyzed by western blotting using an anti-phospho-NF-κB-p65 antibody. (E) For cell migration, cells were pretreated with Bay11-7082 (1 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. Representative phase contrast images are shown for 48 h (n = 3). Data are expressed as mean ± SEM (C) or mean (A, B, D) of three independent experiments (n = 3). * P

    Article Snippet: Actinomycin D, cycloheximide, SB431542, U0126, SB202190, SP600125, helenalin, and Bay11-7082 were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Migration, Zymography, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot

    Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or pretreated with wortmannin or U0126 at the indicated doses for 1 h at 37°C (G) or transfected with or without siRNAs against PI3K p85α or MEK (H) and then incubated with or without PSaV VLPs. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or pretreated with wortmannin or U0126 at the indicated doses for 1 h at 37°C (G) or transfected with or without siRNAs against PI3K p85α or MEK (H) and then incubated with or without PSaV VLPs. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Incubation, Western Blot, Transfection, Infection, Expressing

    Inhibition of the PI3K/Akt and MEK/ERK signaling pathways affects PSaV infectivity and viral protein expression. (A to D) LLC-PK cells were pretreated with noncytotoxic concentrations of wortmannin or U0126 for 1 h at 37°C and then infected with PSaV (MOI of 1 FFU/cell) for 36 h in the presence (A and B) or absence (C and D) of 200 μM GCDCA. (A and C) Levels of PSaV VPg protein were determined by Western blotting. GAPDH was used as a loading control. The intensity of VPg relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane. (B and D) Viral titers were determined by TCID 50 assay. The data are presented as means and standard deviations of the results of three independent experiments. Differences were evaluated using one-way analysis of variance. *, P

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Inhibition of the PI3K/Akt and MEK/ERK signaling pathways affects PSaV infectivity and viral protein expression. (A to D) LLC-PK cells were pretreated with noncytotoxic concentrations of wortmannin or U0126 for 1 h at 37°C and then infected with PSaV (MOI of 1 FFU/cell) for 36 h in the presence (A and B) or absence (C and D) of 200 μM GCDCA. (A and C) Levels of PSaV VPg protein were determined by Western blotting. GAPDH was used as a loading control. The intensity of VPg relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane. (B and D) Viral titers were determined by TCID 50 assay. The data are presented as means and standard deviations of the results of three independent experiments. Differences were evaluated using one-way analysis of variance. *, P

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Inhibition, Infection, Expressing, Western Blot

    PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were inoculated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then infected with or without PSaV in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 min postinoculation (mpi). The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells transfected with scrambled siRNA (Scram) or siRNA against PI3K p85α or MEK were harvested at 24 and 48 h posttransfection. The downregulation of each protein by siRNA knockdown was evaluated by Western blotting using antibodies specific for each protein. GAPDH was used as a loading control. (E) LLC-PK cells transfected with or without each siRNA were incubated with PSaV (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were determined by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were inoculated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then infected with or without PSaV in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 min postinoculation (mpi). The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells transfected with scrambled siRNA (Scram) or siRNA against PI3K p85α or MEK were harvested at 24 and 48 h posttransfection. The downregulation of each protein by siRNA knockdown was evaluated by Western blotting using antibodies specific for each protein. GAPDH was used as a loading control. (E) LLC-PK cells transfected with or without each siRNA were incubated with PSaV (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were determined by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Western Blot, Infection, Expressing, Transfection, Incubation

    Inhibition of PSaV trafficking by blockade of PI3K/Akt and MEK/ERK signaling pathways. (A) LLC-PK cells were incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for the indicated times at 37°C in the presence of 200 μM GCDCA. The cells were then fixed, permeabilized, and further incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2. After incubation with a FITC-conjugated anti-mouse IgG antibody, the cells were processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with the early endosome marker EEA1 or the late endosome marker LAMP2. The boxed areas are magnified and shown under each panel. (B and C) LLC-PK cells were pretreated with or without wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) for 1 h at 37°C and then infected with AF594-labeled PSaV particles (approximately 415 particles per cell) in the presence (B) or absence (C) of 200 μM GCDCA for 3 h. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against EEA1 or LAMP2 and then with a FITC-conjugated secondary antibody to visualize colocalization of AF594-labeled PSaV particles with EEA1 or LAMP2. The boxed areas are magnified and shown under each panel. All experiments were performed in triplicate, and a representative set of results is shown. Bars, 10 μm (A) and 20 μm (B and C). (D and E) Quantification of AF595-labeled PSaV particles colocalized with the early endosome marker EEA1 (D) and the late endosome marker LAMP2 (E) was performed using 10 confocal microscopy images of cells treated under the conditions described above by use of the ImageJ program. Quantification of signals was made with a threshold of 0.03 to 1.3 µm 2 as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Inhibition of PSaV trafficking by blockade of PI3K/Akt and MEK/ERK signaling pathways. (A) LLC-PK cells were incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for the indicated times at 37°C in the presence of 200 μM GCDCA. The cells were then fixed, permeabilized, and further incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2. After incubation with a FITC-conjugated anti-mouse IgG antibody, the cells were processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with the early endosome marker EEA1 or the late endosome marker LAMP2. The boxed areas are magnified and shown under each panel. (B and C) LLC-PK cells were pretreated with or without wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) for 1 h at 37°C and then infected with AF594-labeled PSaV particles (approximately 415 particles per cell) in the presence (B) or absence (C) of 200 μM GCDCA for 3 h. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against EEA1 or LAMP2 and then with a FITC-conjugated secondary antibody to visualize colocalization of AF594-labeled PSaV particles with EEA1 or LAMP2. The boxed areas are magnified and shown under each panel. All experiments were performed in triplicate, and a representative set of results is shown. Bars, 10 μm (A) and 20 μm (B and C). (D and E) Quantification of AF595-labeled PSaV particles colocalized with the early endosome marker EEA1 (D) and the late endosome marker LAMP2 (E) was performed using 10 confocal microscopy images of cells treated under the conditions described above by use of the ImageJ program. Quantification of signals was made with a threshold of 0.03 to 1.3 µm 2 as described in Materials and Methods.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Inhibition, Incubation, Labeling, Marker, Confocal Microscopy, Infection

    Bile acid-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were treated with or without 200 μM GCDCA and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were determined by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells were transfected with or without siRNAs against PI3K p85α or MEK and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Bile acid-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were treated with or without 200 μM GCDCA and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were determined by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells were transfected with or without siRNAs against PI3K p85α or MEK and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Western Blot, Expressing, Transfection

    MEK1 maintains liver CSC self-renewal dependent on SIRT1 Non-CSCs (Huh7-Nanog Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM U0126 or DMSO for 48 hours. Liver CSCs (Huh7-Nanog Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs ( C ) and non-CSCs ( A ) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs ( D ) and non-CSCs ( B ) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. ( E – F ) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = 0.52 × (W) 2 × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.

    Journal: Oncotarget

    Article Title: MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

    doi: 10.18632/oncotarget.7972

    Figure Lengend Snippet: MEK1 maintains liver CSC self-renewal dependent on SIRT1 Non-CSCs (Huh7-Nanog Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM U0126 or DMSO for 48 hours. Liver CSCs (Huh7-Nanog Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs ( C ) and non-CSCs ( A ) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs ( D ) and non-CSCs ( B ) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. ( E – F ) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = 0.52 × (W) 2 × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.

    Article Snippet: Chemical and reagents Inhibitors with the follows: U0126 (662005) were purchased from Merck.

    Techniques: Over Expression, Cell Culture, Staining, Inhibition, Injection, Mouse Assay

    MEK1 signaling activity is required for the maintenance of liver CSC self-renewal ( A ) Huh7-Nanog Pos cells were co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) in sphere-forming conditions for 7 days, counted at the same magnification. ( B ) Huh7-Nanog Pos cells were treated with different concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and grown for 14 days. Cells were stained with crystal violet and counted. ( C ) Western blot analysis of stemness protein expression in Huh7- and PLC/PRF/5-Nanog Pos cells, which co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. ( D ) Huh7-Nanog Pos cells were treated with 5 μM U0126 for 14 days, while the negative control treated with DMSO for 14 days. Then we subcutaneous injected 1 × 10 2 , 1 × 10 3 , 1 × 10 4 cells into NOD-SCID mice. After 30 days, we harvested and counted the tumors. Extreme Limiting Dilution Analysis was acquired from http://bioinf.wehi.edu.au/software/elda/ .

    Journal: Oncotarget

    Article Title: MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

    doi: 10.18632/oncotarget.7972

    Figure Lengend Snippet: MEK1 signaling activity is required for the maintenance of liver CSC self-renewal ( A ) Huh7-Nanog Pos cells were co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) in sphere-forming conditions for 7 days, counted at the same magnification. ( B ) Huh7-Nanog Pos cells were treated with different concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and grown for 14 days. Cells were stained with crystal violet and counted. ( C ) Western blot analysis of stemness protein expression in Huh7- and PLC/PRF/5-Nanog Pos cells, which co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. ( D ) Huh7-Nanog Pos cells were treated with 5 μM U0126 for 14 days, while the negative control treated with DMSO for 14 days. Then we subcutaneous injected 1 × 10 2 , 1 × 10 3 , 1 × 10 4 cells into NOD-SCID mice. After 30 days, we harvested and counted the tumors. Extreme Limiting Dilution Analysis was acquired from http://bioinf.wehi.edu.au/software/elda/ .

    Article Snippet: Chemical and reagents Inhibitors with the follows: U0126 (662005) were purchased from Merck.

    Techniques: Activity Assay, Cell Culture, Staining, Western Blot, Expressing, Planar Chromatography, Negative Control, Injection, Mouse Assay, Software

    MEK1 mainly promotes SIRT1 expression in HCC population ( A ) Western blot analysis the sirtuins expression level in CSCs and non-CSCs, compared with U0126 inhibited CSCs. ( B ) Western blot analysis SIRT1 expression in CSCs after co-cultured with indicated concentration of U0126 or PD98059 ( D ) for 48 hours. ( C ) Western blot analysis SIRT1 expression in CSCs which cultured with 5 μM U0126 for indicated times. ( E ) Western blot analysis SIRT1 expression in CSCs transduced with lentiviruses expressing the indicated shRNA for 48 hours. Those experiments were repeated in two HCC cell lines (Huh7 and PLC/PRF/5).

    Journal: Oncotarget

    Article Title: MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

    doi: 10.18632/oncotarget.7972

    Figure Lengend Snippet: MEK1 mainly promotes SIRT1 expression in HCC population ( A ) Western blot analysis the sirtuins expression level in CSCs and non-CSCs, compared with U0126 inhibited CSCs. ( B ) Western blot analysis SIRT1 expression in CSCs after co-cultured with indicated concentration of U0126 or PD98059 ( D ) for 48 hours. ( C ) Western blot analysis SIRT1 expression in CSCs which cultured with 5 μM U0126 for indicated times. ( E ) Western blot analysis SIRT1 expression in CSCs transduced with lentiviruses expressing the indicated shRNA for 48 hours. Those experiments were repeated in two HCC cell lines (Huh7 and PLC/PRF/5).

    Article Snippet: Chemical and reagents Inhibitors with the follows: U0126 (662005) were purchased from Merck.

    Techniques: Expressing, Western Blot, Cell Culture, Concentration Assay, Transduction, shRNA, Planar Chromatography

    MEK1 inhibitor decreases liver CSCs proliferation ability in vitro ( A ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells under different U0126 concentrations treatment as indicated (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were seeded (1 × 10 3 ) and cultured for another 6 days before analyzed with CCK8. ( B ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells were cultured with or without 5 μM U0126 for 48 hours. Cells were harvested for immunofluorescence (IF) analysis by anti-Ki67 antibodies. Scale bar, 10 μm. ( C ) Cell cycle profiles of 5 μM U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-Nanog Pos cells followed by treatment with Sodium butyrate. Percentage in each histogram indicates the portion of cells remaining in each cell cycle phase.

    Journal: Oncotarget

    Article Title: MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

    doi: 10.18632/oncotarget.7972

    Figure Lengend Snippet: MEK1 inhibitor decreases liver CSCs proliferation ability in vitro ( A ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells under different U0126 concentrations treatment as indicated (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were seeded (1 × 10 3 ) and cultured for another 6 days before analyzed with CCK8. ( B ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells were cultured with or without 5 μM U0126 for 48 hours. Cells were harvested for immunofluorescence (IF) analysis by anti-Ki67 antibodies. Scale bar, 10 μm. ( C ) Cell cycle profiles of 5 μM U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-Nanog Pos cells followed by treatment with Sodium butyrate. Percentage in each histogram indicates the portion of cells remaining in each cell cycle phase.

    Article Snippet: Chemical and reagents Inhibitors with the follows: U0126 (662005) were purchased from Merck.

    Techniques: In Vitro, Planar Chromatography, Cell Culture, Immunofluorescence, Negative Control

    Effects of U0126, oxaliplatin and 5-FU on SW48 cells. Cell viability was measured using CCK-8 assay and is represented as the percentages of the untreated group value. (A) CCK-8 was performed following the treatment of SW48 and NCI-H508 cells with increasing concentrations of U0126 for 72 h. There was a statistical difference between the two groups (*P

    Journal: Molecular Medicine Reports

    Article Title: MEK inhibitor enhanced the antitumor effect of oxaliplatin and 5-fluorouracil in MEK1 Q56P-mutant colorectal cancer cells

    doi: 10.3892/mmr.2018.9730

    Figure Lengend Snippet: Effects of U0126, oxaliplatin and 5-FU on SW48 cells. Cell viability was measured using CCK-8 assay and is represented as the percentages of the untreated group value. (A) CCK-8 was performed following the treatment of SW48 and NCI-H508 cells with increasing concentrations of U0126 for 72 h. There was a statistical difference between the two groups (*P

    Article Snippet: U0126 was purchased from Selleck Chemicals (cat. no. S1102; Houston, TX, USA).

    Techniques: CCK-8 Assay

    Gene expression induced by U0126 combined with or without oxaliplatin/5-FU. (A) ERCC1 mRNA levels in SW48 cells were examined by RT-qPCR following treatment with U0126 and/or oxaliplatin. (B) TYMS mRNA levels in SW48 cells were examined by RT-qPCR following treatment with U0126 and/or 5-FU. The graph depicts the fold change in ERCC1/TYMS levels normalized to β-actin levels. Data are presented as means ± standard deviation. Western blot analysis results of (C) ERCC1 and (D) TYMS protein expression levels in cells treated with U0126 in combination with oxaliplatin or 5-FU, respectively. **P

    Journal: Molecular Medicine Reports

    Article Title: MEK inhibitor enhanced the antitumor effect of oxaliplatin and 5-fluorouracil in MEK1 Q56P-mutant colorectal cancer cells

    doi: 10.3892/mmr.2018.9730

    Figure Lengend Snippet: Gene expression induced by U0126 combined with or without oxaliplatin/5-FU. (A) ERCC1 mRNA levels in SW48 cells were examined by RT-qPCR following treatment with U0126 and/or oxaliplatin. (B) TYMS mRNA levels in SW48 cells were examined by RT-qPCR following treatment with U0126 and/or 5-FU. The graph depicts the fold change in ERCC1/TYMS levels normalized to β-actin levels. Data are presented as means ± standard deviation. Western blot analysis results of (C) ERCC1 and (D) TYMS protein expression levels in cells treated with U0126 in combination with oxaliplatin or 5-FU, respectively. **P

    Article Snippet: U0126 was purchased from Selleck Chemicals (cat. no. S1102; Houston, TX, USA).

    Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Western Blot

    Immunofluorescence staining for γH2AX. (A) γH2AX foci images at a magnification of ×400. Cells were treated with or without 2 µM U0126, combined with oxaliplatin or 5-FU for 72 h. γH2AX expression was detected by immunofluorescence staining and a fluorescence microscope. Cell nuclei were stained with DAPI. (B) Distribution of foci in SW48 cells following exposure to U0126 and/or oxaliplatin/5-FU. **P

    Journal: Molecular Medicine Reports

    Article Title: MEK inhibitor enhanced the antitumor effect of oxaliplatin and 5-fluorouracil in MEK1 Q56P-mutant colorectal cancer cells

    doi: 10.3892/mmr.2018.9730

    Figure Lengend Snippet: Immunofluorescence staining for γH2AX. (A) γH2AX foci images at a magnification of ×400. Cells were treated with or without 2 µM U0126, combined with oxaliplatin or 5-FU for 72 h. γH2AX expression was detected by immunofluorescence staining and a fluorescence microscope. Cell nuclei were stained with DAPI. (B) Distribution of foci in SW48 cells following exposure to U0126 and/or oxaliplatin/5-FU. **P

    Article Snippet: U0126 was purchased from Selleck Chemicals (cat. no. S1102; Houston, TX, USA).

    Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Microscopy

    Effect of 0.01–0.2% sericin ( A ) and 0–20 μM U0126 ( B ) on the phosphorylation of ERK1/2 in HCE-T cells. ( A ) 0.01–0.2% sericin was dissolved in 0.05% DMSO, and treated for 24 h. ( B ) 0–20 μM U0126 was dissolved in 0.05% DMSO, and treated for 24 h. The phosphorylation of ERK1/2 was enhanced with the increase in sericin concentration; 20 μM U0126 prevented the phosphorylation of ERK1/2 in HCE-T cells.

    Journal: International Journal of Molecular Sciences

    Article Title: Instillation of Sericin Enhances Corneal Wound Healing through the ERK Pathway in Rat Debrided Corneal Epithelium

    doi: 10.3390/ijms19041123

    Figure Lengend Snippet: Effect of 0.01–0.2% sericin ( A ) and 0–20 μM U0126 ( B ) on the phosphorylation of ERK1/2 in HCE-T cells. ( A ) 0.01–0.2% sericin was dissolved in 0.05% DMSO, and treated for 24 h. ( B ) 0–20 μM U0126 was dissolved in 0.05% DMSO, and treated for 24 h. The phosphorylation of ERK1/2 was enhanced with the increase in sericin concentration; 20 μM U0126 prevented the phosphorylation of ERK1/2 in HCE-T cells.

    Article Snippet: Materials Pure Sericin™ (30 kDa), U0126 and MedGel® were obtained from Wako Pure Chemical Industries (Osaka, Japan), SCH772984 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

    Techniques: Concentration Assay

    Corneal image ( A ) and corneal wound healing ( B ) in rat eyes treated with sericin and/or U0126. 1% sericin and 200 μM U0126 were dissolved in 0.2% DMSO, and instilled into the eyes of rats five times a day. The corneal images were obtained by a TRC-500X, and corneal wound healing was calculated according to Equation (2). Normal (○), non-instilled rat. vehicle (△), 0.2% DMSO-instilled rat. U0126 (□), U0126-instilled rat, sericin (▲), sericin-instilled rat. Sericin + U0126 (■), sericin-instilled rat treated with U0126. n = 5–8. * 1 p

    Journal: International Journal of Molecular Sciences

    Article Title: Instillation of Sericin Enhances Corneal Wound Healing through the ERK Pathway in Rat Debrided Corneal Epithelium

    doi: 10.3390/ijms19041123

    Figure Lengend Snippet: Corneal image ( A ) and corneal wound healing ( B ) in rat eyes treated with sericin and/or U0126. 1% sericin and 200 μM U0126 were dissolved in 0.2% DMSO, and instilled into the eyes of rats five times a day. The corneal images were obtained by a TRC-500X, and corneal wound healing was calculated according to Equation (2). Normal (○), non-instilled rat. vehicle (△), 0.2% DMSO-instilled rat. U0126 (□), U0126-instilled rat, sericin (▲), sericin-instilled rat. Sericin + U0126 (■), sericin-instilled rat treated with U0126. n = 5–8. * 1 p

    Article Snippet: Materials Pure Sericin™ (30 kDa), U0126 and MedGel® were obtained from Wako Pure Chemical Industries (Osaka, Japan), SCH772984 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

    Techniques:

    Effect of U0126 on the proliferation of HCE-T cells treated with sericin. Twenty micromoles of U0126, 0.5 μM SCH772984, and/or 0.1% sericin were dissolved in 0.05% DMSO, and treated for 24 h. Vehicle, 0.05% DMSO-treated HCE-T cells. U0126, U0126-treated HCE-T cells, SCH772984, SCH772984-treated HCE-T cells. Sericin, sericin-treated HCE-T cells. Sericin + U0126, sericin and U0126 co-treated treated HCE-T cells. Sericin + SCH772984, sericin and SCH772984 co-treated HCE-T cells. n = 7. * 1 p

    Journal: International Journal of Molecular Sciences

    Article Title: Instillation of Sericin Enhances Corneal Wound Healing through the ERK Pathway in Rat Debrided Corneal Epithelium

    doi: 10.3390/ijms19041123

    Figure Lengend Snippet: Effect of U0126 on the proliferation of HCE-T cells treated with sericin. Twenty micromoles of U0126, 0.5 μM SCH772984, and/or 0.1% sericin were dissolved in 0.05% DMSO, and treated for 24 h. Vehicle, 0.05% DMSO-treated HCE-T cells. U0126, U0126-treated HCE-T cells, SCH772984, SCH772984-treated HCE-T cells. Sericin, sericin-treated HCE-T cells. Sericin + U0126, sericin and U0126 co-treated treated HCE-T cells. Sericin + SCH772984, sericin and SCH772984 co-treated HCE-T cells. n = 7. * 1 p

    Article Snippet: Materials Pure Sericin™ (30 kDa), U0126 and MedGel® were obtained from Wako Pure Chemical Industries (Osaka, Japan), SCH772984 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

    Techniques: