Journal: Cell chemical biology

Article Title: TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

doi: 10.1016/j.chembiol.2021.07.014

Figure Lengend Snippet: Figure 4. T3 and sobetirome stimulate and NH-3 blocks phagocytosis by microglia (A–D’) C57BL/6 mouse primary microglia cells in culture were treated with DMSO vehicle (A + A’), 10 nM T3 (B + B’), 1 mM sobetirome (C + C’), or 2 mM NH-3 (D + D’) for 24 h before 2 mm diameter fluorescent beads (100/cell) were introduced 2 h before cells were fixed and stained for CD11B (green). (E) Elongated morphology of an unactivated/ramified microglia cell. (F) Morphology of an activated microglia cell upon treatment with sobetirome (see the supplemental information). (G) Three-dimensional view down three separate axes for visualization of beads inside the uppermost cell in the sobetirome treatment group (image generated using Imaris Section function). (H) Quantification of phagocytosed beads per treatment group. Scale bars, 20 mm (A–D), 10–20 mm (A’–D’), and 8–10 mm (E–G), as noted. Statistical significance was determined by a two-tailed, unpaired t test for comparisons between vehicle and group then were plotted together. Asterisks represent significant difference from vehicle unless otherwise noted: *p % 0.05, **p % 0.01, ***p % 0.001. All graphs show mean ± SEM.

Article Snippet: The cells were then permeabilized with PBS containing 0.05% saponin (saponin was included in all subsequent incubations and washes) and stained for microglial marker CD11b using monoclonal rat anti-mouse CD11b (1:200 dilution, AbD Serotec, #MCA711) in conjunction with Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:400 dilution,Thermo Fisher, A21202).

Techniques: Staining, Generated, Two Tailed Test

Journal: Molecular cancer

Article Title: The circular RNA circDLG1 promotes gastric cancer progression and anti-PD-1 resistance through the regulation of CXCL12 by sponging miR-141-3p.

doi: 10.1186/s12943-021-01475-8

Figure Lengend Snippet: Fig. 4 CircDLG1 promotes tumor progression by increasing the infiltration of MDSCs. a C57BL/6 mice were injected with MFC-sh-NC or MFC-sh-circDLG1 cells, and the tumor volume and tumor metastasis are shown. The mice were sacrificed at 4 weeks after injection. Left: the tumor size was measured, and the tumor volume was calculated every 4 days (n = 10 mice per group), **P < 0.05, two-way ANOVA. Right: the lungs of the mice were removed and paraffin embedded, consecutive sections (4 μm) were made and stained with hematoxylin-eosin, and the micrometastases in the lungs were examined and counted under a dissecting microscope (n = 10 mice per group), **P < 0.05, Student’s t-test. All experiments were repeated three times. Scale bar, left: 5 mm; right: 100 μm. b BALB/c nude mice were injected with MFC-sh-NC or MFC-sh-circDLG1 cells. The mice were sacrificed at 4 weeks after injection. Left: the tumor size was measured, and the tumor volume was calculated every 4 days (n = 10 mice per group). ns, no significance, two-way ANOVA. Right: The lungs of the mice were removed and paraffin embedded. Consecutive sections (4 μm) were made and stained with hematoxylin-eosin, and the micrometastases in the lungs were examined and counted under a dissecting microscope (n = 10 mice per group). ns, no significance, Student’s t-test. All experiments were repeated three times. Scale bar, left: 5 mm; right: 100 μm. c IHC analysis was performed using subcutaneous tumors for CD4, CD8, Gr-1, and CD11b (Gr-1 and CD11b are mouse MDSC markers). HPF, high-power visual field. ns, no significance; **P < 0.05. Student’s t-test. d Flow cytometric analysis of immune cells in subcutaneous tumors. **P < 0.05, Student’s t-test; the experiment was repeated three times. e The expression of CD33 (a marker of human MDSCs) was detected by IHC in human gastric cancer tissues. Scale bar, 50 μm. f The correlation between circDLG1 expression measured by qRT–PCR and CD33+ cells. n = 30, *P < 0.05, Pearson correlation analysis

Article Snippet: The following antibodies were used: anti-mouse IFNγ (BD Biosciences), anti-mouse CD45 (BD Biosciences), anti-mouse Gr-1 (TONBO Biosciences), anti-mouse Ly6G (TONBO Biosciences), anti-mouse ly6C (BD Biosciences), anti-mouse CD11b (TONBO Biosciences), anti-mouse F4/80 (BioLegend), anti-human CD11b (TONBO Biosciences), anti-human CD33 (BD Biosciences), and anti-human HLA-DR (BD Biosciences).

Techniques: Injection, Staining, Microscopy, Expressing, Marker, Quantitative RT-PCR

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 1 Hepatic cholesterol accumulation and M1 macrophages infiltration in MASH. Six-week-old male C57BL/6J mice were fed either a NCD or a CDAHFD for six weeks (n = 3). (a) Body weight gain curves and (b) liver/body weight ratio was recorded. (c) Serum ALT and AST levels were detected. (d) Liver sections stained with H&E and ORO, and immunohistochemically for F4/80+ and CD11b+, 100× magnification. (e-f) Quantifies hepatic TC and TG levels. (g) NAS score. (h-j) Hepatic relative mRNA expression of Tnfa, Il1b and Il6. (k-l) Hepatic relative protein levels of CD86, ARG-1 and CD206. (m-o) Flow cytometry analysis of macrophages (live+CD45+F4/80+CD11b+) and M1 macrophages (live+CD45+F4/80+CD11b+iNOS+). (p-q) Flow cytometry analysis of TREM2 (live+CD45+F4/80+TREM2+). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 between groups

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 3 Correlation between DHCR7 expression and macrophage polarization in MASH. (a) Relative expression of cholesterol biosynthesis and efflux genes in primary macrophages after ox-LDL (100 µg/ mL) or ox-LDL combined with statin treating for 24 h. (b) Expression levels of cholesterol biosyn thesis and efflux genes in primary macrophages polarized to M1 with LPS (100 ng/mL) or M2 with IL-4 (20 ng/mL) for 24 h. (c-d) Western blot analysis for DHCR7 protein in M1 and M2 macrophages, with densitometry. (e) Immunofluorescence staining for DHCR7 in the polarized-macrophages, 200× magnification. (f-g) Multiple immunofluorescence co-localization for DHCR7 and CD11B on livers of NCD or CDAHFD mice. (h-i) DHCR7 protein and mRNA levels in total liver and primary macrophages isolated from livers of mice on control and CDAHFD. (j-k) Immunofluorescence multi-labeling of liver sections from NC and MASH patients for DHCR7 and macrophage marker CD68 to determine the proportion of DHCR7+CD68+ cells among total CD68+ cells. Data are presented as mean ± SD, with n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between groups

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Isolation, Control, Labeling, Marker

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 5 Myeloid-macrophage-specific DHCR7 deficiency exacerbates MASH progression. Wildtype (WT) and myeloid-specific DHCR7 knockout (DHCR7 cKO) mice were fed with a normal control diet (CON) or a CDAHFD for 10 weeks (n = 7). (a) Body weight progression, (b) liver/body weight ratio, and (c-d) serum ALT and AST levels were detected. (e-f) Hepatic TG and TC levels were measured. (g) ELISA quantification of liver IL-1β and IL-6 levels. (h) Histological analysis of liver sections is presented through H&E, ORO staining, and immunohistochemistry for F4/80+ and CD11b+ cells (200× magnification). (i) NAS score. (j-k) Relative protein levels of CD86, ARG-1, CD206 were determined, with densitometry. (l) Flow cytometry of primary macrophage in cKO mice and WT with different diets. (m) Flow cytometry of TREM2 in macrophages. (n) Predominantly of the M1 type in macrophages, including quantification of both the percentage of M1 macrophages and M1 macrophage counts per liver weight. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between group

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Flow Cytometry