mk-0812  (Merck & Co)

Journal: Journal of biochemistry

Article Title: Identification of effective CCR2 inhibitors for cancer therapy using humanized mice.

doi: 10.1093/jb/mvad086

Figure Lengend Snippet: Fig. 1. CCR2 inhibition suppresses lung metastasis of breast cancer cell line. (A) Strategy for generating mouse CCR2 knockout mice by CRISPR/Cas9 electroporation. The target sequence of the guide RNA was designed on the 5′ end side of the mouse CCR2 ORF. RNP was prepared by mixing the CRISPR RNA (Table 1) and transactivating crRNA with recombinant Cas9 protein. Mouse zygotes of the C57BL/6 J strain were subjected to electroporation with RNP. (B) T7E1 assay of mosaic mice obtained by CRISPR/Cas9 electroporation in Figure 1A. The CCR2 ORF regions of mosaic and wild-type mice were amplified by PCR using DNA obtained from the tails, and the PCR products were denatured, reannealed and treated with T7E1 endonuclease I. CCR2 mutations were found in mouse no. 6. (C) Sanger sequencing the mouse CCR2 ORF region in mouse no. 6. (D) Flow cytometry analysis of bone marrow cells recovered from wild-type and CCR2 knockout (KO) mice using anti-mouse CCR2 (Anti-mCCR2) antibody. (E) Flow cytometry analysis of lung M-MDSCs in wild-type and CCR2 KO mice. ∗∗∗P < 0.001 (unpaired two-sided Student’s t-test). (N = 4). (F) Schematic representation of breast cancer cells’ lung metastasis evaluation assay. Wild-type or CCR2 KO mice were injected with tdTomato-labeled E0771 cancer cell line into the tail vein, and 14 days later, lungs were removed and evaluated for tdTomato-positive lung metastases. (G) Lung metastases were evaluated 14 days after tail vein injection of tdTomato-labeled E0771 cancer cell line. Lungs were photographed under blue light irradiation (left). The percentage of tdTomato-positive area in the lung was quantitated with ImageJ software. (right). ∗∗∗P < 0.001 (unpaired two-sided Student’s t-test). Scale bars: 5 mm. (N = 15 for wild-type; N = 9 for CCR2 KO).

Article Snippet: THP-1 cell line (in HBSS with 0.05% BSA) was incubated with Fure 2-AM (final concentration 5.0 μM) for 30 min at 37◦C in the dark, washed twice with HBSS with 0.05% BSA and added each CCR2 inhibitor (MK0812 (Cayman), PF04634817 (Sigma-Aldrich), PF04136309 (PharmaBlock), INCB3344 (Chemscene), cenicriviroc (Axon), JNJ2714149 (R&D systems), CCR2RA-[R] (MedChemExpress), CAS-445479-97-0 (Santa Cruz Biotechnology), RS504393 (Sigma-Aldrich) and RS102895 (Sigma-Aldrich)).

Techniques: Inhibition, Knock-Out, CRISPR, Electroporation, Sequencing, Recombinant, Amplification, Flow Cytometry, Injection, Labeling, Irradiation, Software

Journal: Journal of biochemistry

Article Title: Identification of effective CCR2 inhibitors for cancer therapy using humanized mice.

doi: 10.1093/jb/mvad086

Figure Lengend Snippet: Fig. 3. Generation of human CCR2B knock-in mice. (A) Strategy for generating human CCR2B knock-in ES cells by homologous recombination. The human CCR2B knock-in construct consists of human CCR2B ORF and HA (homologous arm). (B) Genotyping of cloned ES cells by PCR. Knock-in of the human CCR2B allele was confirmed in clone no. 29. (C) Southern blot analysis of DNA from human CCR2B knock-in ES cells (clone no. 29). (D) F0 generation chimeric mice were obtained by microinjection of clone no. 29 into blastocysts. (E) F1 generation mice were obtained by crossing F0 generation chimeric mice with C57BL/6 J mice. (F) Genotyping of F1 generation mice by PCR. N.C., negative control, P.C., positive control. DNA extracted from wild-type C57BL/6 mice was used as N.C. DNA extracted from chimeric mice in Figure 4D as P.C. (G) Flow cytometry analysis of bone marrow cells recovered from wild-type and human CCR2B knock-in (indicated as hCCR2B KI) mice using anti-mouse CCR2 (shown as Anti-mCCR2) or anti-human CCR2 (shown as Anti-hCCR2) antibodies.

Article Snippet: THP-1 cell line (in HBSS with 0.05% BSA) was incubated with Fure 2-AM (final concentration 5.0 μM) for 30 min at 37◦C in the dark, washed twice with HBSS with 0.05% BSA and added each CCR2 inhibitor (MK0812 (Cayman), PF04634817 (Sigma-Aldrich), PF04136309 (PharmaBlock), INCB3344 (Chemscene), cenicriviroc (Axon), JNJ2714149 (R&D systems), CCR2RA-[R] (MedChemExpress), CAS-445479-97-0 (Santa Cruz Biotechnology), RS504393 (Sigma-Aldrich) and RS102895 (Sigma-Aldrich)).

Techniques: Knock-In, Homologous Recombination, Construct, Clone Assay, Southern Blot, Microinjection, Negative Control, Positive Control, Flow Cytometry

Journal: Cells

Article Title: Innate Immune Pathways Promote Oligodendrocyte Progenitor Cell Recruitment to the Injury Site in Adult Zebrafish Brain

doi: 10.3390/cells11030520

Figure Lengend Snippet: Activation of Tlr1/2 and Cxcr3 creates a detrimental environment by inducing oligodendroglia accumulation in a microglia/monocyte-independent manner. ( A ) Scheme of the experimental design to analyze the consequences of double-inhibitor treatment (NBI 74330 and CU CPT22). ( B ) Graph illustrating the size of the injured volume 7 days after skull injury and vehicle, Tlr1/2 inhibitor (CU CPT22), or double Tlr1/2 and Cxcr3 inhibitor (NBI 74330 and CU CPT22) treatment. p -values are based on a one-way ANOVA ( p -value = 1.971 × 10 −4 ) with a post-hoc Dunnett test (Many-to-One). ( C ) Experimental scheme designed to study restorative neurogenesis upon different treatments. ( D , E ) Images depicting HuC/D + and BrdU + cells located in the parenchyma following vehicle ( D ) and double-inhibitor ( E ) treatment. ( F ) Dot-plot showing the total density (whole telencephalon) of HuC/D + and BrdU + after vehicle and Tlr1/2 and Cxcr3 inhibitor treatment. p -value is based on WelcH′s t -test with unequal variances. ( G ) Diagram illustrating the ventricular zone (25 µm from the ventricle surface) and the parenchyma (blue area) in the telencephalic region. Restorative neurogenesis was measured by the proportion of newly generated neurons (HuC/D + and BrdU + ) that migrated towards the parenchyma with respect to the total number (ventricular zone and parenchyma) of new neurons. ( H ) Graph depicting the proportion of HuC/D + and BrdU + cells located in the telencephalic parenchyma after vehicle and Tlr1/2 and Cxcr3 inhibitor treatment. p -value is based on Student’s t -test with equal variances. ( I ) Design of the experimental workflow to analyze the effect of Tlr1/2 and Cxcr3 inhibitors on accumulation of Olig2:GFP + cells after microglia/monocytes depletion. ( J , K ) Micrographs depicting the reactivity of Olig2:GFP + cells after skull injury at 4 dpi with microglia/monocyte depletion and vehicle ( J ) or Tlr1/2 and Cxcr3 inhibitor treatments ( K ). ( L ) Graph illustrating the density of Olig2:GFP + cells at the injury site at 4 dpi following Clodrosome + Ccr2 (MK-0812) inhibitor treatment (microglia/monocyte depletion protocol), Encapsome (empty liposomes, control for Clodrosome; ventricular injection) and Clodrosome + Ccr2 + Tlr1/2 (CU CPT22) + Cxcr3 (NBI 74330) inhibitor treatments. The decrease in Olig2:GFP + cell accumulation after Tlr1/2 and Cxcr3 inhibitor treatment was maintained in microglia/monocyte-depleted brain. p -values are based on a one-way ANOVA ( p -value = 7.957 × 10 −3 ) with a post-hoc Tukey Test (All Pairs). ( M ) Design of the experimental protocol used to analyze injury-induced neurogenesis (BrdU-based birth dating) in microglia/monocyte-depleted brains treated with vehicle or Tlr1/2 and Cxcr3 inhibitor cocktail. ( N , P ) Micrographs of injured telencephala at 7 dpi showing the generation of new neurons (HuC/D + /BrdU + ) after vehicle ( N ) and Tlr1/2 and Cxcr3 inhibitor ( P ) treatment in microglia/monocyte-depleted brains. ( O , O′ , Q , Q′ ) are magnifications of the areas boxed in ( N , P ), respectively. White arrowheads depict double HuC/D + and BrdU + cells. The level of the cross-section is indicated in the inset. ( R ) Graph depicting the proportion of HuC/D + and BrdU + cells located in the telencephalic parenchyma after vehicle and Tlr1/2 and Cxcr3 inhibitor treatment. p -value is based on Student’s t -test with equal variances. All images are full z-projections of a confocal stack. Data are shown as mean ± SEM; each data point represents one animal. Scale bars in ( N , P ) = 100 μm; scale bars in ( D , E , J , K , O , O′ , Q , Q′ ) = 20 μm. Abbreviations: dpi: days post-injury; Veh: vehicle; Inh: inhibitors. Symbol description: red triangle: vehicle; dark blue triangle: Tlr1/2 inhibitor, CU CPT22; light blue triangle: double inhibitors, NBI 74330 and CU CPT22; orange triangle: ventricular Clodrosome injection; purple triangle: intraperitoneal Ccr2 inhibitor injection, MK-0812.

Article Snippet: Ccr2 inhibitor (MK-0812, 82.5 mg/kg, Cayman Chemical, Ann Arbor, MI, USA) was administered by intraperitoneal injection daily, starting 2 days before the injury (Figure 5 and ).

Techniques: Activation Assay, Generated, Liposomes, Control, Injection