Analysis of BRG1 expression in HCC by qRT-PCR and immunohistochemical staining.

<p>(<b>A,B</b>) BRG1 is overexpressed significantly in HCC tissue (n = 13) compared to non-tumour counterpart (n = 10) respectively non-tumour liver tissue of patients not suffering from HCC (n = 13). A more detailed analysis of non-tumour liver tissue revealed no difference of BRG1 expression between tissue of non-fibrosis (n = 13) and fibrosis/cirrhosis (n = 10). Shown are the relative expression levels, non-tumour liver tissue respectively non-fibrosis tissue were standardised as 1. (<b>C</b>) Normal hepatocytes are showing no expression of BRG1. (<b>D</b>) Positive BRG1 staining in HCC tissue. (<b>G</b>) Analysis of BRG1 expression in immunohistochemistry by immunoreactive score. A varying degree of BRG1 expression in HCC was found ranging from a minor score of 1 to a maximum score of 12. The determined scores were evenly distributed. Differences were due to a high variety of intensity as well as the percentage of positive stained cells. Scale bar represents 50μm.</p

BRG1 knockdown impairs colony formation and modulates cyclin family.

<p>(<b>A,B</b>) Colony formation assay was performed to analyse proliferation of both human HCC cell lines HepG2 and HuH7. Colony formation was reduced significantly in both cell lines, HepG2 and HuH7, n = 3. (<b>C</b>) Analysis of mRNA expression by qRT-PCR showed a significant down-regulation of cyclinB and cyclinE expression in BRG1-suppressed HepG2 cells at the time point of 40h after transfection, n = 4. (<b>D</b>) HuH7 cells showed a trend towards lower expression though this was not significant, n = 4. Negative control was standardised as 1.</p

BRG1 knockdown impairs invasiveness and modulates MMP7.

<p>Invasion assay was performed to analyse the role of BRG1 for invasive ability of human HCC cell lines. (<b>A-E</b>) In both cell lines down-regulated BRG1 expression impaired invasive ability. (<b>F</b>) Analysis of mRNA levels after down-regulation of BRG1 showed a significant decrease of MMP7 expression for both cell lines 40h after transfection. Negative control was standardised as 1. Scale bar represents 100μm.</p

Immunohistochemical staining of BRG1 in HCC.

<p><b>(A-J)</b> BRG1 is expressed in HCC tissue <b>(A,C,E,G,I)</b> but not in non-tumour tissue counterpart <b>(B,D,F,H,J)</b>. <b>(A,C,E,G,I)</b> HCC tissue is showing different expression levels of BRG1 due to a high variety of intensity as well as the percentage of positive stained cells. Scale bar represents 50μm.</p

BRG1 knockdown impairs proliferation.

<p>(<b>A,B</b>) Human HCC cell lines HepG2 and HuH7 showed expression of BRG1 on mRNA and protein level. (<b>A</b>) After transfection, a highly significant down-regulation of mRNA levels of BRG1 was achieved for both HepG2 (P<0.001) and HuH7 (P<0.001) cell lines, n = 4. (<b>B</b>) Analysed by Western Blot, protein expression was also decreased in HepG2 and HuH7 cell lines after transfection, n = 3. Proliferation was analysed by growth curves for both human HCC cell lines HepG2 and HuH7. (<b>C,D</b>) Growth curves, n = 3 and (<b>E,F</b>) BRG1 expression at different time points after transfection, n = 3. Growth curves of HepG2 (<b>C</b>) and HuH7 (<b>D</b>) cells revealed a significant decrease of proliferation as long as BRG1 is suppressed significantly (<b>E,F</b>). 10 days after transfection proliferation rates began to equalize (<b>C,D</b>) and siRNA targeting BRG1 started to lose its impact (<b>E,F</b>). (<b>E,F</b>) Negative control was standardised as 1.</p

Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions

<div><p>Background</p><p>Activation of the immune system in terms of subseptic conditions during liver regeneration is of paramount clinical importance. However, little is known about molecular mechanisms and their mediators that control hepatocyte proliferation. We sought to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy (PH), and to uncover the impact of the integrin lymphocyte function-associated antigen-1 (LFA-1) on liver regeneration in a subseptic setting.</p><p>Methods</p><p>Wild-type (WT) and LFA-1^-/- mice underwent a 2/3 PH and low-dose lipopolysaccharid (LPS) application. Hepatocyte proliferation, immune cell infiltration, and cytokine profile in the liver parenchyma were determined.</p><p>Results</p><p>Low-dose LPS application after PH results in a significant delay of liver regeneration between 48h and 72h, which is associated with a reduced number of CD3⁺ cells within the regenerating liver. In absence of LFA-1, an impaired regenerative capacity was observed under low-dose LPS application. Analysis of different leukocyte subpopulations showed less CD3⁺NK1.1⁺ NKT cells in the liver parenchyma of LFA-1^-/- mice after PH and LPS application compared to WT controls, while CD3^-NK1.1⁺ NK cells markedly increased. Concordantly with this observation, lower levels of NKT cell related cytokines IL-12 and IL-23 were expressed in the regenerating liver of LFA-1^-/- mice, while the expression of NK cell-associated CCL5 and IL-10 was increased compared to WT mice.</p><p>Conclusion</p><p>A subseptic situation negatively alters hepatocyte proliferation. Within this scenario, we suggest an important impact of NKT cells and postulate a critical function for LFA-1 during processes of liver regeneration.</p></div

Absence of LFA-1 results in decreased numbers of NKT cells within the regenerating liver.

<p>Detection of T cells within the liver parenchyma of WT or LFA-1^-/- mice after PH and LPS application. (A) CD3⁺ cells within the liver were quantified on at least 5 sections per group and number of CD3⁺ T cells per mm² tissue was calculated. Data from 3 mice per time point are shown. Statistical differences between WT and LFA-1^-/- were tested by two-way ANOVA-test. (B) Representative images 48h and 7d (168h) post-PH and LPS are depicted using immunofluorescence staining of liver cryosections. CD3⁺ T cells appear red and nuclear DAPI blue. Scale bars indicate 100μm. (C, D) Detection of CD3⁺NK1.1^- T cells, CD3^-NK1.1⁺ NK cells or CD3⁺NK1.1⁺ NKT cells in the regenerating liver of WT or LFA-1^-/- mice at distinct time points after PH and low-dose LPS using flow cytometry. (C) A representative dot-plot is depicted for gated CD45⁺ leukocytes in a WT or a LFA-1^-/- liver 24h post-PH and LPS. (D) Quantitative analysis of CD3⁺NK1.1⁺ NKT cells and CD3^-NK1.1⁺ NK cells in the liver parenchyma of WT and LFA-1^-/- mice. Data show percentage of cells from all leukocytes within the liver parenchyma (mean ± SD) from 4 mice per time point. The significances refer to the difference between WT and the LFA-1^-/- mice and were tested by two-way ANOVA.</p

Characterization of immunocompetent cells in the regenerating liver.

<p>Number of Gr1⁺ (A) or CD3⁺ (C) immune cells per mm² liver parenchyma were quantified in the regenerating liver at distinct time points after PH, PH plus LPS application or sham-procedure plus LPS application in WT mice. The 0h timepoint represents liver tissue of WT mice (n = 2) without any treatment and before the operation (preoperative situation). Representative images for Gr1⁺ (B, green) or CD3⁺ (D, red) cells within the liver parenchyma 6h after sham procedure plus LPS, PH plus LPS or PH only in WT mice are shown. At least 5 sections per time point and treatment were analyzed. Each value represents the mean ± SD from 3 independent mice per time point. Statistical significances were analyzed by two-way ANOVA-test and refer to the comparison between PH and PH and LPS application. Scale bars indicate 50μm. Gr1⁺ cells in green, CD3⁺ cells in red, nuclei in blue (DAPI).</p

LPS dose-response profile and liver regeneration under low-dose LPS stimulus.

<p>WT mice underwent sham-operation or PH followed by intraperitoneal application of different doses of LPS. LPS dosage per 20g body weight is depicted. (A) Quantitative analysis of the liver specific enzyme alanine-aminotransferase (ALT) 24h post surgery (n = 3). (B) ALT serum levels in WT mice 24h after sham-operation or PH in absence or presence of 50μg LPS per 20g body weight are shown (n = 3). (C) Liver/body-weight-ratio in percent of WT mice at different time points upon PH or PH and LPS application. Seven days (168h) post surgery complete liver regeneration was observed. Graph represents mean ± SD from 3 mice for each setting. (D) Hepatocyte proliferation within the liver parenchyma at distinct time points post-PH or PH and LPS. Proliferating hepatocytes were detected by Ki-67 (green) and nuclear DAPI (blue) using immunofluorescence staining of liver cryosections. Liver sections of non-operated mice served as untreated control. Representative images are shown (n = 3 mice). Scale bars indicate 100μm. (E) Percentage of Ki-67 positive hepatocytes in relation to all hepatocytes was quantified. Mean ± SD from at least 5 cryosections for each time point from two mice (n≥2000 hepatocytes) is shown.</p

Impact of LFA-1 on liver regeneration.

<p>(A) Liver/body-weight-ratio in percent and amount of ALT in the serum of WT or LFA-1^-/- mice at different time points upon PH and application of 50μg LPS per 20g body weight. Values represent mean ± SD from 3 mice per time point. Statistical significances were analyzed by two-way ANOVA-test and refer to the comparison between the WT and LFA-1^-/- mice. (B) Detection of proliferating Ki67-positive hepatocytes in the liver parenchyma at 48h and 7 days in the liver parenchyma of WT or LFA-1^-/- mice. Scale bars indicate 100μm. Ki-67 (green), nuclear DAPI (blue). (C) Mean ± SD of Ki-67 positive hepatocytes in relation to all hepatocytes in percent is shown. At least 5 cryosections for each time point from two mice (n≥2000 hepatocytes) were analyzed. (D) Analysis of TNFα-mRNA upon PH and low-dose LPS in the liver parenchyma of WT and LFA-1^-/- mice. Values represent mean ± SD from 3 mice per time point. Statistical differences between WT and LFA-1^-/- were tested by two-way ANOVA-test. (E) Quantification of Gr1⁺ neutrophils in the liver parenchyma after PH and LPS application by immunofluorescence staining on liver cryosections. Values represent mean ± SD from at least 5 cryosections for each time point (n = 3 mice). Statistical differences between the WT and LFA-1^-/- were tested by two-way ANOVA-test.</p