Journal: Science Advances

Article Title: Compromised macrophages contribute to progression of MASH to hepatocellular carcinoma in FGF21KO mice

doi: 10.1126/sciadv.ado9311

Figure Lengend Snippet: ( A ) Protein levels of Alox15 by Western blotting in the liver tissues of WT/FGF21KO mice with WSHFD/CD diets. ( B ) Alox15 mRNA levels by qPCR analysis in the isolated hepatocytes and KCs and the protein levels of Alox15 by Western blotting in KCs from the WT/FGF21KO mice with WSHFD/CD diets. ( C ) Immunofluorescent staining using the antibodies of anti-ALOX15 and anti-F4/80 to detect the positive ALOX15 macrophages. Green, ALOX15-positive staining cells; red, F4/80-positive staining cells; blue, 4′,6-diamidino-2-phenylindole (DAPI) staining to detect the nuclei as a counterstain. ( D ) Protein levels of FAS and cleaved caspase-3 by Western blotting in the isolated mouse KCs challenged with 13-HODE and treated with N -acetyl- l -cysteine (NAC). ( E ) Protein levels of FAS and cleaved caspase-3 by Western blotting in the liver tissues from FGF21KO-MASH mice with rhFGF21 treatment compared to vehicle controls or WT-CD controls. ( F ) Histology-based NAFLD active score (NAS) in the WT/ALOX15KO mice with HFMCD/WSHFD diets. ( G ) CyTOF analysis in the isolated macrophages from mice to detect the KCs defined as F4/80 hi Tim4 + CX3CR1 − cells and the MoMFs defined as CD11b + Tim4 − CX3CR1 + cells in KC pool. The FGF21KO-MASH mice were treated daily with PD146176 or rhFGF21 for 4 weeks. ( H ) Analysis of the RNA-seq data for the mRNA fold changes of immune response signaling pathway and cytokine-cytokine interaction signaling. 21KO, FGF21KO. * P < 0.05 and ** P < 0.01.

Article Snippet: Two MASH model were established by feeding (i) HFMCD (A06071302, l -amino acid diet with 60% kcal fat, 0.1% methionine, and no added choline, Research Diet, Inc., NJ) or (ii) WSHFD (D16030909, 60% kcal fat and 19% kcal fructose, Research Diets Inc., NJ).

Techniques: Western Blot, Isolation, Staining, RNA Sequencing

Journal: Science Advances

Article Title: Compromised macrophages contribute to progression of MASH to hepatocellular carcinoma in FGF21KO mice

doi: 10.1126/sciadv.ado9311

Figure Lengend Snippet: ( A ) Schematic diagram for establishing MASH-HCC models and the gross anatomy of tumor mass in four MASH-HCC models with CD, HFMCD, WSHFD, and HFD, respectively. ( B ) Maximal diameter of tumor nodules and number of tumor nodules in four MASH-HCC models. ( C ) Histology-based NAS in the MASH-HCC mice with HFMCD and WSHFD diets. ( D ) Schematic diagram for rhFGF21 treatment in the MASH-HCC mice with HFMCD and WSHFD diets. ( E ) CyTOF analysis of the KCs (F4/80 hi Tim4 + CX3CR1 − cells) and MoMFs (CD11b + Tim4 − CX3CR1 + cells) from the MASH mice and MASH-HCC mice, as well as the control mice. ( F ) LCFAs in the FGF21KO-MASH-HCC mice compared to normal WT controls. ( G ) Protein levels of cPLA2, Alox15, and MAGL by Western blotting in the liver tissues of FGF21KO-MASH-HCC mice compared to normal WT controls. ( H ) The serum FFA levels in FGF21KO-HCC mice in comparison with the WT-HCC mice, with three diets (CD, HFMCD, and WSHFD). ( I ) S1P levels in serum and liver tissue from FGF21KO-HCC mice in comparison with the WT-HCC mice, with three diets (CD, HFMCD, and WSHFD). 21KO, FGF21KO. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Two MASH model were established by feeding (i) HFMCD (A06071302, l -amino acid diet with 60% kcal fat, 0.1% methionine, and no added choline, Research Diet, Inc., NJ) or (ii) WSHFD (D16030909, 60% kcal fat and 19% kcal fructose, Research Diets Inc., NJ).

Techniques: Control, Western Blot, Comparison

Journal: Science Advances

Article Title: Compromised macrophages contribute to progression of MASH to hepatocellular carcinoma in FGF21KO mice

doi: 10.1126/sciadv.ado9311

Figure Lengend Snippet: The major signaling components mediated alteration of KC homeostasis and carcinogenetic transition polarization mediated NSAH-HCC transition being studied. HFMCD, high-fat methionine/choline-deficient diet; WSHFD, Western-style diet (high-fat and high-fructose); M1, macrophage M1 polarization; M2, macrophage M2 polarization; TAM, tumor-associated macrophage.

Article Snippet: Two MASH model were established by feeding (i) HFMCD (A06071302, l -amino acid diet with 60% kcal fat, 0.1% methionine, and no added choline, Research Diet, Inc., NJ) or (ii) WSHFD (D16030909, 60% kcal fat and 19% kcal fructose, Research Diets Inc., NJ).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Inhibition of Extracellular Vesicle-Associated MMP2 Abrogates Intercellular Transfer of Hepatic miR-122 to Tissue Macrophages and Curtails Liver Inflammation

doi: 10.1101/2021.01.04.425217

Figure Lengend Snippet: A Isolation and characterization of Non-Parenchymal Cells (NPCs) from chow diet and MCD diet fed mouse liver. Schematic diagram showing isolation and FACS sorting of NPCs from chow diet fed (Control) vs MCD diet fed mice livers with CD11b (APC) and F4/80 (FITC) gating. B Haematoxylin and Eosin stained micrographs of control and MCD diet fed mice liver sections C-D Hepatic immune cells in MCD diet fed mice show significantly higher levels of internalized miR-122 with respect to control. Relative internalized miR-122 level in sorted RHM cells is shown (C, top panel). As a control, levels of pre-miR-122 (C, middle panel) and internalized miR-16 (C, bottom panel) have also been measured. Relative levels of internalized miR-122 in sorted KC cells is shown (D-top panel, left side). As a control, relative pre-miR-122 (D-bottom panel, left side) and internalized miR-16 (D-top panel, right side) and miR-155 levels (D-bottom panel, right side) have also been shown in the sorted KC cells. miR-155 induction is characteristic of inflammatory response. E-F Higher levels of internalized miR-122 correlates with increased expression of various pro-inflammatory cytokine mRNAs. Levels of pro-inflammatory cytokines (TNFα, IL-6 and IL1β) in sorted RHM cells (E) and KC cells (F) from control and MCD diet fed mice livers are shown. All mRNA and miRNA detections were done by RT-qPCR. RT-qPCR detection of cytokine mRNAs and pre-miR-122 was done using specific primers from 200 ng of cellular RNA. RT-qPCR detection of internalized miRNAs and miR-155 was done from 25ng of isolated cellular RNA. Normalization of miR-122, miR-155 and miR-16 were done against U6 snRNA. Normalization of precursor miR-122 and cytokine mRNAs were performed in respect to 18S rRNA levels. Data represents Mean ± SD. All images are representative of atleast 4 independent mice from each group. For statistical significance, minimum three independent experiments were considered in each case unless otherwise mentioned and error bars are represented as mean ± S.D. P-values were calculated by utilising Student’s t-test. ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.0001.

Article Snippet: Mice were randomly divided into two groups and fed either standard chow diet or methionine and choline deficient diet MCD (MP Biomedicals; #0296043910) up to four weeks.

Techniques: Isolation, Staining, Expressing, Quantitative RT-PCR