immortalized hcecs b4g12 cells  (Creative Bioarray Inc)

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Effect of interleukin 1β (IL-1β) stimulation on human corneal endothelial cells (CEC). (A) Treatment with IL-1β resulted in increased expression of interleukin 1 receptor 1 (IL-1R1). (B) Treatment of human CEC with IL-1β resulted in expression of fibroblast growth factor-2 (FGF2); phosphorylation of Akt (p-Akt), inhibitor κB kinase (p-IKK) and p38 (p-p38); and decrease in inhibitor κB (IκB) levels. This effect could be attenuated in a dose-dependent manner using interleukin 1 receptor antagonist (IL-1Ra). (C) Treatment of human CEC with IL-1β or FGF2 resulted in enhanced cell migration as measured using scratch-induced directional migration assay. Co-treatment with SU5402, a pan FGF signaling inhibitor, abolished the IL-1β (* p = 0.005) and FGF2 (** p = 0.002) induced migration in human CEC. Co-treatment of human CEC with IL-1Ra also abolished the IL-1β induced migration (+ p = 0.2, ++ p < 0.001). Data represent the mean ± S.D. of three independent experiments. (D) Treatment of human CEC with IL-1β resulted in activation of activator protein-1 (AP-1) and nuclear factor κB (NF-κB). Activation of AP-1 and NF-κB by IL-1β could be blocked with IL-1Ra in human CEC (* p < 0.001, ** p < 0.001). SFM, serum free media; SU, SU5402; C, control.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Expressing, Phospho-proteomics, Migration, Activation Assay, Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Effect of interleukin receptor-associated kinase (IRAK) 1/4 inhibition on interleukin 1β (IL-1β)-stimulated human corneal endothelial cells (CEC). (A) Fibroblast growth factor-2 (FGF2) starved human CEC were pretreated with IRAK 1/4 inhibitor (IRAKi) for 2 h and then treated with IL-1β for 10 min. The IL-1β ± IRAKi treated human CEC were maintained in serum-free medium (SFM) for indicated times. Pretreatment with IRAKi blocked IL-1β dependent phosphorylation of Akt (p-Akt), inhibitor κB kinase(p-IKK), and p38 (p-p38). (B) Nuclear fractions were prepared from FGF2-starved human CEC pretreated with IRAKi for 2 h during FGF2 starvation before stimulation with IL-1β for 10 min followed by 4 h incubation in SFM. Lamin B was used to normalize the nuclear protein concentration in activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) activity assays (data not shown). Pretreatment with IRAKi blocked both AP-1 and NF-κB activities in IL-1β stimulated human CEC. Data represent the mean ± S.D. of three independent experiments (* p < 0.001, ** p < 0.001). (C) Treatment with IRAKi blocked the IL-1β dependent migration of human CEC (*** p < 0.001). Data represent the mean ± S.D. of three independent experiments. IRAKi, IRAK 1/4 inhibitor; C, positive control.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Inhibition, Phospho-proteomics, Incubation, Protein Concentration, Activity Assay, Migration, Positive Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Effect of phosphatidyl inositol (PI) 3-kinase inhibition on interleukin-1β (IL-1β) stimulated human corneal endothelial cells (CEC). (A) Cell lysates were prepared from fibroblast growth factor-2 (FGF2) starved cells pretreated with PI 3-kinase inhibitor LY294002 for 2 h and then treated with IL-1β for 10 min followed by incubation in serum-free medium (SFM) for indicated times. Pretreatment with LY294002 resulted in decreased FGF2 expression along with decreased phosphorylation of Akt (p-Akt), inhibitor κB kinase (p-IKK), and p38 (p-p38) in IL-1β stimulated human CEC. (B) Cell lysates were prepared from human CEC treated as described in (A). Pretreatment with LY294002 did not interfere with interleukin receptor-associated kinase (IRAK) 1 and tumor necrosis factor receptor associated factor (TRAF) 6 interaction in IL-1β stimulated human CEC. (C) Nuclear fractions were prepared from FGF2-starved human CEC pretreated LY294002 for 2 h during FGF2 starvation before stimulation with IL-1β for 10 min followed by 4 h incubation in SFM. Lamin B was used to normalize the nuclear protein concentration in activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) activity assays (data not shown). Pretreatment with LY294002 blocked both AP-1 and NF-κB activities in IL-1β stimulated human CEC. Data represent the mean ± S.D. of three independent experiments (* p < 0.001, ** p < 0.001). (D) Pretreatment with LY294002 abolished the IL-1β induced migration in human CEC (*** p = 0.005). Data represent the mean ± S.D. of three independent experiments. IP, immunoprecipitation; IB, immunoblotting; LY, LY294002; C, positive control.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Inhibition, Incubation, Expressing, Phospho-proteomics, Protein Concentration, Activity Assay, Migration, Immunoprecipitation, Western Blot, Positive Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Effect of activator protein-1 (AP-1) inhibition on interleukin-1β (IL-1β) stimulated human corneal endothelial cells (CEC). (A) Cell lysates prepared from FGF2-starved cells pretreated with SB203580, an AP-1 inhibitor, for 2 h and then treated with IL-1β for 10 min followed by incubation in serum-free medium (SFM) for indicated times. Pretreatment with SB203580 resulted in decreased fibroblast growth factor-2 (FGF2) expression along with decreased phosphorylation of p38 (p-p38) in IL-1β stimulated human CEC. (B) Pretreatment with SB203580 had no effect on inhibitor κB kinase phosphorylation (p-IKK) in IL-1β stimulated human CEC. (C) Nuclear fractions were prepared from FGF2-starved human CEC that were pretreated with SB203580 for 2 hours during FGF2 starvation, followed by stimulation with IL-1B for 10 min and then incubated with SFM for 4 h. Lamin B was used to normalize the nuclear protein concentration in AP-1 and nuclear factor-κB (NF-κB) activity assays (data not shown). Pretreatment with SB203580 blocked AP-1 but not NF-κB activities in IL-1β stimulated human CEC. Data represent the mean ± S.D. of three independent experiments (* p < 0.001, ** p = 0.264). (D) Pretreatment with SB203580 decreased but did not completely block IL-1β enhanced migration in human CEC (*** p = 0.03). Data represent the mean ± S.D. of three independent experiments. SB, SB203580; LY, LY294002; C, positive control.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Inhibition, Incubation, Expressing, Phospho-proteomics, Protein Concentration, Activity Assay, Blocking Assay, Migration, Positive Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Effect of nuclear factor -κB (NF-κB) inhibition on interleukin 1β (IL-1β) stimulated human corneal endothelial cells (CEC). (A) Cell lysates prepared from serum-starved cells pretreated with sulfasalazine, a NF-κB inhibitor, for 2 h and then treated with IL-1β for 10 min followed by incubation in serum-free medium (SFM) for indicated times. Pretreatment with sulfasalazine resulted in decreased FGF2 expression along with decreased phosphorylation of p65 (p-p65) in IL-1β stimulated human CEC. (B) Pretreatment with sulfasalazine had no effect on p38 phosphorylation (p-p38) following IL-1β stimulation in human CEC. (C) Nuclear fractions were prepared from FGF2-starved human CEC that were pretreated with sulfasalazine for 2 hours during FGF2 starvation, followed by stimulation with IL-1B for 10 min and then incubated with SFM for 4 h.Lamin B was used to normalize the nuclear protein concentration in activator protein-1 (AP-1) and NF-κB activity assays (data not shown). Pretreatment with sulfasalazine blocked NF-κB but not AP-1 activities in IL-1β stimulated human CEC. Data represent the mean ± S.D. of three independent experiments (* p = 0.31, ** p < 0.001). (D) Pretreatment with sulfasalazine decreased but did not completely block IL-1β enhanced migration in human CEC (*** p = 0.054). Data represent the mean ± S.D. of three independent experiments. Sul, sulfasalazine; C, positive control.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Inhibition, Incubation, Expressing, Phospho-proteomics, Protein Concentration, Activity Assay, Blocking Assay, Migration, Positive Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Effect of simultaneous activator protein-1 (AP-1) and nuclear factor -κB (NF-B) inhibition on interleukin 1β(IL-1β) stimulated human corneal endothelial cells (CEC).(A) Cell lysates prepared from FGF2-starved cells were pretreated with SB203580 and sulfasalazine for 2 h and then treated with IL-1β for 10 min followed by incubation in serum-free medium (SFM) for indicated times. Pretreatment with SB203580 and sulfasalazine resulted in severely decreased fibroblast growth factor 2 (FGF2) expression along with decreased phosphorylation of p38 (p-p38) and p65 (p-p65) in IL-1β stimulated human CEC. (B) Nuclear fractions were prepared from FGF2-starved human CEC that were pretreated with SB203580 and sulfasalazine for 2 hours during FGF2 starvation, followed by stimulation with IL-1B for 10 min and then incubated with SFM for 4 h. Lamin B was used to normalize the nuclear protein concentration in activator protein (AP-1) and nuclear factor-κB (NF-κB) activity assays (data not shown).Pretreatment with SB203580 and sulfasalazine blocked both AP-1 and NF-κB activities in human CEC that were stimulated by IL-1β (* p < 0.001, ** p < 0.001). Data represent the mean ± S.D. of three independent experiments. (C) Pretreatment with SB203580 and sulfasalazine completely blocked the IL-1β enhanced migration in human CEC (*** p = 0.001). Data represent the mean ± S.D. of three independent experiments. C, positive control; SB, SB203580; Sul, sulfasalazine.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Inhibition, Incubation, Expressing, Phospho-proteomics, Protein Concentration, Activity Assay, Migration, Positive Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Binding of activator protein-1 (AP-1) and nuclear factor -κB (NF-κB) to the FGF2 promoter in human corneal endothelial cells (CEC). (A) Schematic illustration of putative AP-1 and NF-κB binding sites in the human fibroblast growth factor-2 (FGF2) promoter. The DNA sequence that is different from conserved NF-κB binding site (GGGATTTCCC) is italicized. The location of each primer used in ChIP assays is indicated.(B) FGF2-starved human CEC were pretreated with either SB203580 or sulfasalazine for 2 h and then stimulated with IL-1β for 10 min, followed by incubation in serum-free medium (SFM) for 4 h. At the end of treatment, cross-linked cell lysates were treated with nuclease and immunoprecipitated with an anti-NF-κB antibody. The purified DNA samples were subjected to PCR using the shown primer sets. The expected PCR product sizes are: NF1-NF3, 180 bp; NF1-NF4, 260 bp; NF2-NF3, 120 bp; NF2-NF4, 200 bp. Simulation with interleukin-1β (IL-1β) resulted in NF-κB binding to the FGF2 promoter in human CEC. Pretreatment with sulfasalazine inhibited NF-κB binding to the promoter while SB203580 pretreatment had no effect on NF-κB binding. (C) The cross-linked cell lysates from SB203580 pretreated human CEC were treated with nuclease and immunoprecipitated with an anti-AP-1 antibody. The purified DNA samples were subjected to PCR using the shown primer sets. The expected PCR product sizes are: AP1-AP5, 282 bp; AP2-AP5, 234 bp; AP3-AP5, 180 bp; AP4-AP5, 156 bp. IL-1β stimulation resulted in AP-1 binding to the FGF2 promoter in human CEC. Pretreatment with SB203580 inhibited AP-1 binding to the promoter while sulfasalazine pretreatment had no effect on AP-1 binding. Anti-histone H3 antibody was used for positive control (+) and normal rabbit IgG was used for negative control (-) in the ChIP assays. SB, SB203580; Sul, sulfasalazine.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Binding Assay, Sequencing, Incubation, Immunoprecipitation, Purification, Positive Control, Negative Control

Journal: Biology of the cell / under the auspices of the European Cell Biology Organization

Article Title: Interleukin-1? enhances cell migration through AP-1 and NF-?B pathway dependent FGF2 expression in human corneal endothelial cells

doi: 10.1111/boc.201200077

Figure Lengend Snippet: Schematic presentation of the pathway for interleukin-1β (IL-1β) induced human corneal endothelial cell (CEC) migration that is mediated by fibroblast growth factor-2 (FGF2) expression dependent on parallel activator protein-1 (AP-1) and nuclear factor -κB (NF-κB) activation. Binding of IL-1β to interleukin 1 receptor 1 results in assembly of the canonical signaling components, including interleukin receptor-associated kinase (IRAK) 1/4 and tumor necrosis factor receptor associated factor (TRAF) 6, that in turn activates PI 3-kinase. This leads to parallel activation of NF-κB and AP-1 resulting in FGF2 expression and enhanced human CEC migration.

Article Snippet: Cell culture Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (Valtink et al., 2007; Götze et al., 2008 ).

Techniques: Migration, Expressing, Activation Assay, Binding Assay