GSK-3 is involved in the increase in ANTXR expression induced by ET. (A and B) RAW 264.7 cells were pretreated with 20 mM LiCl for 2 h, followed by a 6-h treatment with 100 ng/ml PA plus 100 ng/ml EF (ET) or no treatment (NT). (A) Following treatment, RNA was isolated and Antxr2 mRNA levels were measured from 50 ng total RNA by RT-qPCR. Values represent the average relative Antxr2 mRNA levels from three independent experiments performed in triplicate ± SD. (B) Following treatment, cells were harvested, stained with 80 nM AFPA, and analyzed by flow cytometry. Values represent the average MFI from three independent experiments ± SD. (C) RAW 264.7 cells were pretreated with 0.05% DMSO or 5 μM BIO for 2 h, followed by a 16-h treatment with 1 μg/ml PA plus 1 μg/ml EF (ET) or no treatment (NT). Following treatment, cells were harvested, stained with 80 nM AFPA, and analyzed by flow cytometry. Values represent the average MFI from three independent experiments ± SD. (A to C) Statistical significance was calculated using one-way ANOVA with Bonferroni's multiple-comparison test. *, P < 0.05; ***, P < 0.001 (compared with control). (D and E) RAW 264.7 cells were transfected with siRNA as described in Materials and Methods. (D) Forty-eight hours after transfection, WCE were generated, and WCE (10 μg/sample) were analyzed by Western blotting using antibodies specific for GSK-3β or GAPDH. The bar graph represents densitometry analysis of GSK-3β levels with and without siRNA against GSK-3α/β. The values are the relative band intensities (%) for GSK-3β from 3 experiments ± SD. (E) Twenty-four hours after transfection, the cells were exposed to 1 μg/ml PA plus 1 μg/ml EF (ET) for 20 h. Following treatment, cells were harvested, stained with 80 nM AFPA, and analyzed by flow cytometry. Data represent the relative MFI (%) of PA binding averaged from 6 independent experiments ± SD. (D and E) Statistical significance was calculated using two-tailed Student's t test. **, P < 0.01; ***, P < 0.001.

Interactions between GSK-3 and CREB. (A and B) RAW-Neg, RAW-CRE, or RAW-CMV cells were treated for 24 h with 1 μg/ml PA plus 1 μg/ml EF (ET) or untreated (NT), with or without 20 mM LiCl or 5 μM BIO. Following treatment, cell extracts were generated and a luciferase assay was performed. (A) The bar graph indicates the average amount of CRE-dependent luciferase produced under each condition after normalizing the data with luciferase produced from the RAW-Neg cells. Values represent normalized average RFU from RAW-CRE cells from three independent experiments ± SD. Statistical significance between samples treated with ET only and samples treated with ET plus inhibitors was calculated using two-tailed Student's t test. *, P < 0.05; ***, P < 0.001. (B) Values represent normalized average RFU from RAW-CMV cells from two independent experiments ± SD. (C) RAW 264.7 cells were transfected with siRNA as described in Materials and Methods and, 24 h later, were exposed to 1 μg/ml PA plus 1 μg/ml EF (ET) for 6 h. Following treatment, cell extracts were generated and a luciferase assay was performed. Values represent normalized average RFU from RAW-CRE cells from three independent experiments ± SD. Statistical significance was determined as described for panel A. **, P < 0.01. (D) RAW 264.7 cells were treated with 1 μg/ml PA plus 1 μg/ml EF (ET) for 6 h in the presence or absence of 20 mM LiCl or 5 μM BIO. WCE (10 μg/sample) were analyzed by Western blotting using antibodies against pSer-133 CREB, pSer-129 CREB, total CREB, or GAPDH. (E) The bar graph represents the densitometry analysis of the bands corresponding to pSer-129 CREB when normalized to GAPDH levels. The values are the relative band intensities (%) for pSer-129 CREB from 3 experiments ± SD. Statistical significance between samples treated with ET only and samples treated with ET plus inhibitors was calculated using two-tailed Student's t test. *, P < 0.05; **, P < 0.001.

GSK-3 is required for MDDC maturation and chemotaxis. (A and B) With or without a 2-h pretreatment with LiCl, MDDCs were left untreated (NT), treated with 100 ng/ml LPS, or treated with 100 ng/ml PA plus 100 ng/ml EF (ET). To remove residual endotoxin, untreated MDDCs and ET-treated MDDCs were preincubated with 10 μg/ml polymyxin B for 2 h. (A) For cell surface phenotype, cells were harvested 48 h after treatment and incubated with anti-DC-SIGN-FITC, anti-CD83-APC, and anti-CD86-FITC, followed by flow cytometric analysis. Data represent the individual mean fluorescence intensity (MFI) values for each of five donors. (B) Following a 48-h treatment, migration of treated MDDCs toward MIP-3β was measured using a Transwell assay employing uncoated inserts (5-μm pore). Data are shown as fold migration compared with that for immature (no treatment, NT) MDDCs for each donor and are the average values ± standard deviations (SD) for five donors. (C) RAW 264.7 cells were treated with 1 μg/ml PA plus 1 μg/ml EF (ET) or left untreated (NT) for 6 h with or without 20 mM LiCl, 5 μM BIO, or 0.05% dimethyl sulfoxide (DMSO). RNA was then isolated, and levels of Sdc1 mRNA were determined by reverse transcriptase quantitative PCR (RT-qPCR). Values represent the average relative Sdc1 mRNA levels from three independent experiments performed in triplicate ± SD. (A to C) Statistical significance was calculated using one-way analysis of variance (ANOVA) with Bonferroni's multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with appropriate control).

ACREB inhibits increase in ANTXR expression induced by ET. (A) RAW-CRE and RAW-NF-κB cells were transduced with MIGR1 or ACREB virus as described in Materials and Methods. Forty hours after transduction, cells were treated for 6 h with either 500 ng/ml PA plus 500 ng/ml EF (ET) or 500 ng/ml LPS or left untreated (NT), as indicated. Following treatment, cell extracts were generated and a luciferase assay was performed. The values from the luciferase assay represent normalized average relative fluorescence units (RFU) from three independent experiments performed in triplicate ± SD. (B and C) RAW 264.7 cells transduced with either MIGR1 or ACREB virus were treated for 6 h with 100 ng/ml PA plus 100 ng/ml EF (ET) or not treated (NT). (B) Following treatment, RNA was isolated and Antxr2 mRNA levels were measured from 50 ng total RNA by RT-qPCR. Values represent the average relative Antxr2 mRNA levels from three independent experiments performed in triplicate ± SD. (C) Following treatment, cells were harvested, stained with 80 nM AFPA, and analyzed by flow cytometry. Values represent the average MFI from three independent experiments ± SD. (A to C) Statistical significance was calculated using one-way ANOVA with Bonferroni's multiple-comparison test. **, P < 0.01; ***, P < 0.001.