Hepatic gene induction in murine bone marrow after hepatectomy Shintaro Yamazaki, Kenji Miki, Tadatoshi Takayama, Kiyoshi Hasegawa, Masataka Sata, Yutaka Midorikawa, Hiroyuki Aburatani, Masatoshi Makuuchi  Journal of Hepatology  Volume 44, Issue 2, Pages 325-333 (February 2006) DOI: 10.1016/j.jhep.2005.07.025 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Detecting variant AFP. (a) Schematic representation of authentic and variant AFPs transcripts reported by Kubota et al. and positions of primers used for RT-PCR. (b) RT-PCR results of three AFP primer pairs. AFP sequences including exon 1 (AFP-2 and AFP-3) were similarly amplified compared to the common sequence (AFP-1). Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Effect of AAF on hepatocyte proliferation. Proliferation of hepatocytes 2d after hepatectomy was evaluated using anti-Ki67 labelling. About 70% of the hepatocytes in the hepatectomised mice (PHx group) were positive for Ki67, whereas only 15% were positive in the mice also treated with AAF (AAF/PHx group). Scale bars: 50μm. Original magnifications: ×200. Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Quantitative RT-PCR analysis of whole bone marrow. Comparison of the mRNA levels of several genes in the bone marrow of control, PHx, AAF and AAF/PHx mice (n=5 in each group). Values are expressed relative to the level of mRNA in the control group (mean±standard error (SE); *P<0.05). Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 (a) Time course of AFP expression in bone marrow after hepatectomy. Values are expressed relative to the baseline level of mRNA in the PHx group (mean±SE). (b) AFP mRNA expression in peripheral blood. AFP mRNA was only detected in peripheral blood on day 2 after hepatectomy and only in the AAF/PHx mice. Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 (a) FACS analysis of cells expressing AFP mRNA. Bone marrow cells from AAF/PHx mice 2d after hepatectomy were isolated with FACS using cell-surface markers. AFP mRNA in the positive and negative fraction of each marker was tested. Cells (105) in the positive and negative fractions for each cell-surface marker were collected using FACS after gating-out contaminating lineage-positive cells. Percentages of positive and negative fractions of Lin- cells are listed. (b) AFP mRNA in fractionated bone marrow cells. AFP mRNA was detected in the Lin- and either the CD34+, c-kit+, Sca-1+, CD49f+ or the CD45+ cell population. Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 (a) The hierarchical clustering of 42 genes strongly induced on day 2 in PHx liver compared to AAF/PHx liver. Data are presented in a matrix format. Each row represents a single gene and each column represents an experimental sample. The ratio of the expression of each gene to the mean expression in six samples is represented by a color in the corresponding sample within the matrix. Green squares represent transcript levels below the mean value and red squares represent those above the mean value. Genes marked with asterisks are known to be related to cell division. (b) The hierarchical clustering of 26 genes induced on day 2 in AAF/PHx liver compared with PHx liver. Genes marked with asterisks are known to be induced by TNF or IL-6. The expression levels of IL-6 and TNF are also given in the matrix. Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 (a) The hierarchical clustering of 42 genes strongly induced on day 2 in PHx liver compared to AAF/PHx liver. Data are presented in a matrix format. Each row represents a single gene and each column represents an experimental sample. The ratio of the expression of each gene to the mean expression in six samples is represented by a color in the corresponding sample within the matrix. Green squares represent transcript levels below the mean value and red squares represent those above the mean value. Genes marked with asterisks are known to be related to cell division. (b) The hierarchical clustering of 26 genes induced on day 2 in AAF/PHx liver compared with PHx liver. Genes marked with asterisks are known to be induced by TNF or IL-6. The expression levels of IL-6 and TNF are also given in the matrix. Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Inhibition of endoderm-specific gene expression in bone marrow by dexamethasone in the AAF/PHx model. The levels of mRNA for several genes were compared in the bone marrow of AAF-treated mice without hepatectomy (AAF), with hepatectomy (AAF/PHx) and in AAF/PHx mice administered dexamethasone (AAF/PHx/DEX) (n=5 in each group). Values are expressed relative to the baseline level in the AAF group (mean±SE). Journal of Hepatology 2006 44, 325-333DOI: (10.1016/j.jhep.2005.07.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions