(I) ChIP assays were performed using antibodies of H3K4me1, H3K4me3 and DNMT1 in lysates of AML12 and Hepa1-6 cells. Figure S3. The relationship of miR-144 and miR-451a expression in HCC (= 125). Figure S4. The expression of miR-144 and miR-451a is repressed in human and mouse HCC cell lines and tissues. (A-D) The expression of miR-144 and miR-451a was measured via qRT-PCR in normal hepatocyte (NH) and HCC cell lines Azoramide of human (A and B) and mouse (C, D) organs (= 6). (E, F) Hepa1-6 cells were inoculated intrahepatically into C57BL/6 mice, and paratumor Azoramide and HCC tissues were isolated to measure the expression of miR-144 and miR-451a (= 5). Bars, means SEMs; **, 0.01; ***, 0.001. Figure S5. miR-144 or miR-451a overexpression suppresses HCC development in vivo. (A, B) Control Hepa1-6 cells or those overexpressing miR-144 or miR-451a were inoculated intrahepatically into C57BL/6 mice. Body weight and liver weight were examined three weeks after the injection (= Azoramide 6) (A). Tumors were then dissected, and the expression of miR-144/miR-451a was measured via q-RT-PCR (= 6) (B). (C, D) H22 cells were infected with control or miR-144/miR-451a-overexpressing lentiviruses and were subcutaneously inoculated on C57BL/6 mice. Tumor tissues were excised and Mouse monoclonal to CD11a.4A122 reacts with CD11a, a 180 kDa molecule. CD11a is the a chain of the leukocyte function associated antigen-1 (LFA-1a), and is expressed on all leukocytes including T and B cells, monocytes, and granulocytes, but is absent on non-hematopoietic tissue and human platelets. CD11/CD18 (LFA-1), a member of the integrin subfamily, is a leukocyte adhesion receptor that is essential for cell-to-cell contact, such as lymphocyte adhesion, NK and T-cell cytolysis, and T-cell proliferation. CD11/CD18 is also involved in the interaction of leucocytes with endothelium weighed three weeks after the inoculation (= 5). The expression of miR-144/miR-451a was measured via q-RT-PCR (= = 5) (D). Bars, means SEMs; **, 0.01; ***, 0.001. Figure S6. miR-144 or miR-451a had no effect on proliferation and apoptosis of HCC cells. Hepa1-6 cells were infected by control or miR-144/miR-451a-overexpressing lentiviruses. Cells were then subject to CCK-8 assay (A, = 5), FACS assay for cell cycle pregression (B, = 5) and apoptosis (C, = 5). The expressions of epithelial and mesenchymal markers were also examined via qRT-PCR (D, = 5). Bars, means SEM. Figure S7. miR-144/miR-451a overexpression affects microenvironmental cell subsets in an orthotopic HCC model. (A-E) Control Hepa1-6 cells or those overexpressing miR-144 or miR-451a were inoculated intrahepatically into C57BL/6 mice. Tumors were then dissected, and FACS was performed to analyze the subsets of lymphocytes (A-D) and MDSCs (E) (= 6). Bars, means SEMs; **, 0.01; ***, 0.001. Figure S8. miR-144/miR-451a overexpression modulates the phenotypes of microenvironmental cells in an orthotopic HCC model derived from H22 cells. (A-D) H22 cells were infected with control or miR-144/miR-451a-overexpressing lentiviruses and were subcutaneously inoculated on C57BL/6 mice. The tumor tissues were excised, and FACS was performed to analyze the subsets and differentiation of TAMs (A) and phenotype of lymphocytes (B) from different groups, and the cell percentages were plotted and compared Azoramide (C) (= 5). The mRNA levels of macrophage polarization markers were detected using qRT-PCR in sorted TAMs (D, = 5). Bars, means SEMs; *, 0.05; **, 0.01; ***, 0.001. Figure S9. The miR-144/miR-451a cluster correlates with M1-polarization of microenvironmental macrophages. (A, B) The tumor tissues of HCC patients were stained with F4/80 and p65 (A) and analyzed for expression of indicated markers (B) (1: = 25; 2: = 10; 3: = 10; 4: = 25). Figure S10. miR-144/miR-451a modulates the HCC paracrine function to promote macrophage M1-like polarization and antitumor effects = 6), Azoramide ELISA (B, = 6) and Western blot (D, n =4) assays for the expression of the indicated genes or measurement of NO generation (C, = 8). (E-G) BMDMs were incubated in the supernatant of Hepa1-6 cells as described in (A) and then.