mk-0812 Search Results


ccr2 inhibitor  (MedChemExpress)
93
MedChemExpress ccr2 inhibitor
Fig. 1. <t>CCR2</t> 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).
Ccr2 Inhibitor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mk0812  (TargetMol)
93
TargetMol mk0812
Fig. 1. <t>CCR2</t> 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).
Mk0812, supplied by TargetMol, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ccr2 antagonist mk0812  (MedChemExpress)
94
MedChemExpress ccr2 antagonist mk0812
Fig. 1. <t>CCR2</t> 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).
Ccr2 Antagonist Mk0812, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mk-0812  (Merck & Co)
90
Merck & Co mk-0812
Fig. 1. <t>CCR2</t> 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).
Mk 0812, supplied by Merck & Co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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d-erythro-pentitol derivative mk-0812  (Haihang Industry Co Ltd)
90
Haihang Industry Co Ltd d-erythro-pentitol derivative mk-0812
Fig. 1. <t>CCR2</t> 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).
D Erythro Pentitol Derivative Mk 0812, supplied by Haihang Industry Co Ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mk-0812  (Incyte corporation)
90
Incyte corporation mk-0812
Fig. 1. <t>CCR2</t> 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).
Mk 0812, supplied by Incyte corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mk-0812  (Cayman Chemical)
90
Cayman Chemical mk-0812
Fig. 1. <t>CCR2</t> 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).
Mk 0812, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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orthosteric antagonist mk-0812  (Boehringer Ingelheim)
90
Boehringer Ingelheim orthosteric antagonist mk-0812
Fig. 1. <t>CCR2</t> 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).
Orthosteric Antagonist Mk 0812, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mk-0812  (Adooq Bioscience LLC)
90
Adooq Bioscience LLC mk-0812
Fig. 1. <t>CCR2</t> 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).
Mk 0812, supplied by Adooq Bioscience LLC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mk-0812  (Merck KGaA)
90
Merck KGaA mk-0812
Fig. 1. <t>CCR2</t> 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).
Mk 0812, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


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).

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

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.

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