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. Author manuscript; available in PMC: 2024 Jul 22.
Published in final edited form as: Hepatology. 2023 Nov 17;78(6):1727–1741. doi: 10.1002/hep.32797

FIGURE 6.

FIGURE 6

Spp1 mediates MDA-9–induced macrophage migration. (A) Expression analysis of Spp1 mRNA by Q-RT-PCR in WT and Alb/MDA-9 hepatocytes. Data represent mean ± SEM. *p < 0.01. (B) Analysis of SPP1 protein in the conditioned medium (CM) of WT and Alb/MDA-9 hepatocytes (left panel). Equal protein loading is shown by ponceau S staining (right panel). (C) Gene Expression Profiling Interactive Analysis (GEPIA) showing positive correlation between MDA-9/SDCBP and SPP1 mRNA levels in TCGA liver cancer database. (D) Alb/MDA-9 hepatocytes were treated for 48 h with either BMS-345541 (5 μm; left panel), Cpd 22 (2.5 μm; middle panel), or control and Ilk siRNAs (right panel), and Spp1 mRNA levels were analyzed by Q-RT-PCR. Data represent mean ± SEM. *p < 0.01. (E) Schematic representation of the experimental protocol of macrophage transwell migration assay. WT macrophages (macro) were plated in the top chamber. The bottom chamber contained either CM, CM from WT hepatocytes or CM from Alb/MDA-9 hepatocytes, untreated (UT) or treated with control, or Spp1 siRNA. (F) Representative photomicrographs of macrophage migration assay. (G) Quantification of macrophage migration assay. Data represent mean ± SEM. *p < 0.01 between WT CM and Alb/MDA-9 CM, either UT or treated with control siRNA; #p < 0.01 between Alb/MDA-9 CM, either UT or treated with control siRNA, and Alb/MDA-9 CM treated with Spp1 siRNA. (H) Quantification of macrophage migration assay upon treatment with BMS-345541 (5 μm). Data represent mean ± SEM. *p < 0.01 between WT CM and Alb/MDA-9 CM, either UT or treated with DMSO; #p < 0.01 between Alb/MDA-9 CM, either UT or treated with DMSO, and Alb/MDA-9 CM treated with BMS-345541. Alb, albumin; Con-si, control siRNA; DMSO, dimethyl sulfoxide; Ilk-si, Ilk siRNA; mRNA, messenger RNA; MDA-9, Melanoma differentiation associated gene-9; Q-RT-PCR, quantitative real time polymerase chain reaction; SDCBP, syndecan binding protein; siRNA, small interfering RNA; Spp1, Secreted phosphoprotein 1; TCGA, The Cancer Genome Atlas; TG, transgenic; TPM, transcript count per million; Veh, vehicle; WT, wild-type.