immortalized hcecs b4g12  (Creative Bioarray Inc)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin‐1β

doi: 10.1111/jcmm.15307

Figure Lengend Snippet: Corneal stromal cell‐derived IL‐1β and CCL20 are involved in LPA‐induced proliferation of corneal endothelial cells (CECs). A, Mitomycin C‐pretreated human corneal stromal cells (HCSCs) were maintained in TC medium with or without 20 μmol/L LPA for 2 d before CM collection. ELISA showed that the concentrations of secreted IL‐1β and CCL20 were both significantly greater in the LPA‐treated group. B, The dose‐dependence of IL‐1β‐ and CCL20‐induced B4G12 cell proliferation was determined by cell counts. The number of cells was significantly higher after treatment of recombinant IL‐1β alone for 2 d. In contrast, no difference was observed in the CCL20‐treated group. C, The involvement of IL‐1β in LPA‐induced proliferation of CECs was investigated via neutralization assay of the CM. Immortalized HCECs (B4G12) co‐cultured with mitomycin C‐pretreated HCSCs in TC medium for 5 d were greater in cell number in the LPA‐treated group (20 μmol/L). Subsequent IL‐1β neutralization with 2 ng/mL IL‐1β‐specific antibody (IL‐1β Ab, clone AS10) significantly attenuated the LPA‐induced proliferation in co‐cultures. D, The proliferation‐promoting effect of IL‐1β on RCECs was also examined 2 d later. The fraction of RCECs in the co‐culture increased significantly with increasing concentrations of IL‐1β (20, 200 or 1000 pg/mL). E, The morphology and ECD of the 200 pg/mL IL‐1β‐treated tissue‐cultured rabbit corneal endothelium was examined via immunostaining for ZO‐1 on Day 2. The corneal ECD was significantly higher in the LPA‐treated cells than that in controls and had the normal hexagonal phenotype typical of RCECs. (n = 3; * P < .5, ** P < .05)

Article Snippet: Immortalized HCECs (B4G12) were purchased from Creative Bioarray (NY) and cultured in B4G12 medium (HESFM supplemented with 2% FBS and 10 ng/mL b‐FGF.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Neutralization, Cell Culture, Co-Culture Assay, Immunostaining

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