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: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: The EP2 receptor induces expression of inflammatory enzymes and cytokines in mouse peritoneal macrophages. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, values are mean ± SEM. A, EP2 immunostaining is detected in wild-type but not EP2−/− C57BL/6 primary peritoneal macrophages costained for Cd11b. Scale bar, 100 μm. B, Peritoneal macrophages were stimulated with LPS (10 ng/ml) for 1 and 6 h. qPCR demonstrates a rapid upregulation of EP2 receptor mRNA by 1 h and subsequent downregulation by 6 h following LPS stimulation (n = 4–6 per group; two-way ANOVA for effect of time ##p < 0.01, and effect of interaction p = 0.02; Bonferroni's multiple-comparison tests comparing mean of 1 h vehicle (veh) and 1 h LPS *p < 0.05). C, Peritoneal macrophages were stimulated with LPS (10 ng/ml) +/− the EP2 agonist butaprost (1 μm) or vehicle. qPCR demonstrates an induction of proinflammatory mediators COX-2, iNOS, and gp91phox with LPS stimulation that is further enhanced by costimulation with butaprost (time points 1 h for COX-2 and 6 h for iNOS and gp91phox; n = 4–6 per group; two-way ANOVA for effect of LPS #p < 0.05, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost *p < 0.05, **p < 0.01). D, LPS-induced macrophage NO release is increased by EP2 receptor activation with butaprost (1 μm), whereas EP2−/− macrophages show reduced NO levels compared with EP2+/+ macrophages (n = 4 per group; Student's tests #p < 0.05, ##p < 0.01 comparing EP2+/+ to EP2−/−). E, qPCR demonstrates induction of IL-6 mRNA at 1 h and IL1β mRNA at 6 h, and a trend of decreased TNFα mRNA at 6 h in LPS-stimulated macrophages with addition of butaprost (n = 4–6 per group; two-way ANOVA for effect of LPS ##p < 0.01, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost **p < 0.01 for IL-6 at 1 h and IL1β at 6 h).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Expressing, Immunostaining, Comparison, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Conditional deletion of the EP2 receptor in macrophages suppresses oxidative enzyme and cytokine gene expression. A, Genomic DNA PCR is shown for EP2+/+, EP2lox/+, and EP2lox/lox C57BL/6 mice. B, Quantitative genomic PCR was assayed for EP2+/+ wild-type, EP2+/−, EP2−/−, Cd11bCre;EP2lox/lox, Cd11bCre;EP2lox/+, and Cd11bCre;EP2+/+; values are relative to wild-type EP2+/+ control DNA. C, Peritoneal macrophages were isolated from adult Cd11bCre;EP2lox/lox and Cd11bCre;EP2+/+ mice and sorted using Cd11b antibody-conjugated magnetic beads before qPCR analysis. Basal levels of EP2 mRNA, assayed by qPCR, are reduced by 91% in Cd11bCre;EP2lox/lox compared with control macrophages (left; Cd11bCre;EP2lox/lox vs Cd11bCre;EP2+/+) and are reduced by 56% in LPS-stimulated Cd11bCre;EP2lox/lox macrophages (right; **p < 0.01; n = 5–6 per group). D, Conditional deletion of EP2 in macrophages reduces LPS-mediated increases in proinflammatory gene expression (two-way ANOVA for effect of LPS treatment is represented by ###p < 0. 001; Bonferroni's multiple-comparisons tests comparing mean of Cd11bCre;EP2+/+/LPS and Cd11bCre;EP2lox/lox/LPS were ***p < 0.001; n = 5–6 per group).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Gene Expression, Control, Isolation, Magnetic Beads

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Examination of levels of monocytic populations in Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice. Splenocytes and peripheral blood immune cells were isolated from 3-month-old mice. A, Representative plots for CD11b+ cells gated on CD115 and Ly6C yielding four populations of cells, including CD115−/Ly6C− macrophages, CD115−/Ly6Cint-hi neutrophils, CD115int/Ly6Cint resident monocytes, and CD115hi-int/Ly6Chi inflammatory monocytes in vehicle and LPS-treated mice. B, Quantification of levels of monocytic populations, including macrophages, resident monocytes, and inflammatory monocytes does not show differences between genotypes in vehicle or LPS-treated mice. Levels of neutrophils are decreased in peripheral blood with LPS, but not vehicle stimulation (*p < 0.05; n = 4 mice per group). C, Quantification of CD11b-positive microglia derived from brains of Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice does not show differences in number (n = 5–7 mice per genotype). D, Comparison of copy number of EP2/copy number of 18S is shown for adult microglia and peritoneal macrophages from Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice (n = 4–6 per group; p < 0.05 unpaired t test). Macrophage expression of EP2 in Cd11bCre;EP2+/+ mice was 28-fold higher; however, the percentage reduction of expression with conditional deletion of EP2 was similar in both microglia and macrophages, and was 62.2 and 62.1%, respectively.

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Isolation, Derivative Assay, Comparison, Expressing