Increased expression levels of pIKKβ(S181), pS6K1(T389), and VEGF-A in liver tissues of HBx transgenic mice.

<p>(A). A gross view of representative liver tumors (T1, T2, T3) developed in HBx transgenic mice in several months of breeding. The ALT values are shown. (B). The H&E staining of non-tumor and tumor parts in HBx transgenic mice. (C). The expression levels of pIKKβ (S181), IKKβ, pS6K1 (T389), S6K1, VEGF-A, HBx, and β-actin detected by Western blotting in non-tumor and tumor parts of liver tissues of three HBx transgenic mice (#824, #825, and #826) were compared to the normal liver tissues of the wild-type age-matched mouse. The HBx mRNA levels were also measured by RT-PCR and the GAPDH mRNA levels were used as an internal control. The relative levels of pIKKβ (S181), pS6K1 (T389), and VEGF-A were quantified by densitometry and normalized with total IKKβ, total S6K1, and actin. Results are shown as ratios of average levels of pIKKβ (S181) pS6K1 (T389), and VEGF-A in non-tumor and tumor parts of liver tissues of three HBx transgenic mice (#824, #825, and #826) relative to that in the normal liver tissues of the wild-type age-matched mouse (set as 1). Data are shown as means ± S.D. of measurements of three mice. (D). Immunohistochemistry analyses show expression levels of pIKKβ (S181), pS6K1 (T389), VEGF-A, and CD31 in normal liver tissues of the wild-type mouse, and non-tumor and tumor parts of liver tissues of HBx transgenic mice. One representative data are shown. N = 3.</p

The IKKβ/TSC1/mTOR signaling pathway is activated by HBx.

<p>(A). Expression of HBx mRNA in Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells was detected using semi-quantitative RT-PCR. Levels of GAPDH mRNA were used as an internal control. RNAs of a HBV-positive patient’s serum (P) and RNAs of a control HBV-negative serum (N) were used as controls. (B). Levels of HBx protein were detected in lysates of Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells using Western blotting by antibody specific against HBx protein and β-actin. (C). Levels of pIKKβ(S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin were assessed in lysates of Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells using Western blotting by specific antibody as indicated. (D). Data shown are ratios of viable cells in Hep3Bx and HepG2x cells relative to that in Hep3B and HepG2 cells (set as 1), respectively, at 24 h after seeding using MTT assay. (E). Levels of pIKKβ (S181), pS6K1 (T389), total IKKβ, total S6K1, HBx, and β-actin were assessed in lysates of Huh7 cells transfected with empty vector alone, payw1.2WT, or payw*7. (F). Levels of pIKKβ (S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin were assessed in lysates of Hep3B and Hep3Bx with or without TNF-α treatment using Western blotting as described earlier.</p

Expression of VEGF is increased in Hep3Bx and HepG2x cells and is further enhanced by TNF-α and blocked by IKKβ inhibitor Bay 11-7082 or the mTOR inhibitor rapamycin.

<p>(A). The expression levels of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, and HepG2x cells were measured by ELISA assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041931#s2" target="_blank">Methods</a>. (B). The expression levels of VEGFA mRNA were assessed in Hep3B, Hep3Bx, HepG2, and HepG2x cells using semi-quantitative RT-PCR (left) or real-time RT-PCR (right) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041931#s2" target="_blank">Methods</a>. (C). The amounts of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, or HepG2x cells treated with or without TNF-α in the presence or absence of Bay11-7082 or rapamycin were measured by ELISA assay. Data are shown as means ± S.D. of three experiments. Comparisons were made between different groups as indicated. *<i>P</i><0.001 is determined by X test.</p

Positive association between pIKKβ(S181), pTSC1(S511), and pS6K1(T389) in HBV-associated human HCC specimens.

<p>(A). Immunohistochemistry analysis of pIKKβ (S181), pTSC1(S511) and pS6K1(T389) in tumor tissues of 95 human HBV-associated HCC specimens. Results of one representative specimens stained by specific antibodies are shown. (B). Upper graph shows percentages of specimens with low or high pIKKβ (S181) expression in which pS6K1 (T389) expression was high or was not observed (low). Lower graph shows percentages of specimens with low or high pIKKβ (S181) expression in which pTSC1 (S511) expression was high or was not observed (low). Positive correlations was noted between pIKKβ (S181) and pS6K1 (T389) (*<i>P</i><0.01) and between pIKKβ (S181) and pTSC1 (S511) (*<i>P</i><0.01)<b>.</b> (C) The Kaplan-Meier disease-free survival curves show that expression of pIKKβ (S181) (<i>p</i> = 0.003), pTSC1 (S511) (<i>p</i> = 0.048), or pS6K1 (T389) (<i>p</i> = 0.0027) is associated with early tumor recurrence. Co-expression of pIKKβ (S181) and pS6K1 (T389) (<i>p</i> = 0.0013) was a better predictor of patients’ recurrence-free time survival in HCC patients who received curative surgery for up to 48-month investigation.</p

TNF-α-stimulated increases of pTSC1 (S511), pS6K1 (T389) and cell proliferation in Hep3Bx and HepG2x cells are blocked by the IKKβ inhibitor Bay 11-7082, siRNA specific for IKKβ, and the mTOR inhibitor rapamycin.

<p>(A). Lysates of Hep3Bx and HepG2x cells treated with or without TNF-α in the presence or absence of Bay 11-7082 were analyzed for levels of pIKKβ (S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin using Western blotting as described earlier. (B). Lysates of Hep3Bx and HepG2x cells with transfection of IKKβsiRNAs or control siRNAs were assessed for levels of pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin. (C). Lysates of Hep3Bx and HepG2x cells treated with or without TNF-α in the presence or absence of rapamycin were analyzed for levels of pIKKβ (S181), pS6K1 (T389), total IKKβ, and total S6K1. (D). Data shown are ratios of viable cells in Hep3B, Hep3Bx, HepG2, and HepG2x cells treated with or without TNF-α in the presence or absence of Bay11-7082 or rapamycin relative to that in Hep3B and HepG2 cells without any treatment (set as 1), at 24 h after seeding using MTT assay. Data are shown as means ± S.D. of three experiments. Comparisons were made between different groups as indicated. *P<0.001 is determined by X test.</p

Expression of CLEC4F during embryogenesis.

<p>Yolk sac, embryo and fetal liver in various embryonic stages were collected for CLEC4F detection by (A) qRT-PCR and (B) Western blot. Actin was used as internal control. (C) F4/80 and CLEC4F immunohistochemistry of fetal liver from E14.5 and E17.5, respectively.</p

<i>Clec4f−/−</i> mice showed a similar response of α-GalCer induced activation to wild-type mice <i>in vivo</i>.

<p>Both wild-type and <i>Clec4f−/−</i> littermates were treated with α-GalCer (1 µg/mouse) intravenously. Serum levels of (A) ALT and (B) cytokines, including IL-4 and IFN-γ were analyzed for samples taken at indicated time points after α-GalCer challenge. Data are representative of three independent experiments with similar results. (3 mice in each group).</p

Glycan binding profile of Fc.CLEC4F.

<p><b>Fc.CLEC4F (5 µg/ml) in binding buffer was applied onto glycan array slides.</b> The “Average Binding Intensity” and “Standard Deviation (SD)” among eight glycan replicates were analyzed by ArrayVision software (GE Healthcare).</p

CLEC4F⁺ cells were appeared in the liver environment under Kupffer cell depletion and inflammatory stage.

<p>(A) Kupffer cells were depleted by Cl₂MBP-encapsulated liposome by intravenous injection (100 µl/mouse) at day 0 and livers were harvest at day 1, 7, 14 and 28. F4/80 and CLEC4F immunohistochemistry of liver sections were performed. (B) The numbers of F4/80⁺ or CLEC4F⁺ cells in livers were shown. For generating inflammatory stage, wild-type and <i>Clec4f−/−</i> littermates were infected with <i>L. monocytogenes</i> (1×10⁵ CFU/mouse) intravenously. (C) The numbers of F4/80⁺ or CLEC4F⁺ cells in livers during <i>L. monocytogenes</i> infection. (D) Immunohistochemistry of <i>L. monocytogenes</i> infected livers of wild-type and <i>Clec4f−/−</i> mice at day 5 after infection. (E) Kaplan-Meier survival curves were shown for <i>Clec4f−/−</i> or wild-type littermates with <i>L. monocytogenes</i> infection. The <i>p</i> value was determined by Log-rank test.</p

CLEC4F is involved in the presentation of α-GalCer to NKT cells.

<p>(A) The schematic structure of α-GalCer. (B) The binding curves were obtained from the function of Fc.CLEC4F concentration and fluorescence intensity determined from array images. (C) Competition experiment between solution and surface for Fc.CLEC4F binding to α-GalCer and three derivatives. At different concentration of the competitors, binding curves were obtained from the bound Fc.CLEC4F concentration and fluorescence. (D) Secretion of IFN-γ and IL-4 of NKT cells after incubation of α-GalCer presented by Kupffer cells isolated from wild-type and <i>Clec4f−/−</i> mice. Kupffer cells (1×10⁵ cells) isolated form wild-type and <i>Clec4f−/−</i> littermates were treated with serial concentration of α-GalCer (10, 30, 100, 300 and 1000 ng/ml) then co-cultured with NKT cells (1×10⁵ cells). The supernatant were collected at 72 h post-stimulation and detected the IL-4 and IFN-γ production by ELISA. (E) Effect of CD1d-blocking antibody in the α-GalCer presentation by wild-type and <i>Clec4f−/−</i> Kupffer cells. Kupffer cells were pretreated with CD1d blocking antibody. Three hours later, α-GalCer (300 ng/ml) was added and co-cultured with NKT cells. The supernatant were collected at 72 h post-stimulation and detected the IL-4 and IFN-γ production by ELISA.</p