Journal: Journal of Bone Oncology

Article Title: The metastatic role of the CXCL10-CXCR3 axis and its therapeutic potential in osteosarcoma

doi: 10.1016/j.jbo.2025.100690

Figure Lengend Snippet: The effects of CXCL10 on OS cell migration. A, CXCR3 immunoblotting of seven OS cell lines maintained in DMEM supplemented with 10 % FBS. Vinculin was used as a loading control. B, Transwell migration assays for 3 OS cell lines in response to various concentrations of CXCR3 ligands (CXCL4, CXCL9, CXCL10, and CXCL11) over 4 to 6 h. The chemotactic index (CI) was calculated as the ratio of migrated cells in the CXCL10-stimulated condition to those in the unstimulated condition, n = 3. Statistical significance was determined using one-tailed one-sample t-tests with a reference value of one; n = at least three independent experiments; * P < 0.05; ns = not significant.

Article Snippet: The primary antibodies used were: CXCR3 (1:1000, PA5-28741, Invitrogen), Vinculin (1:5000, V9131, Sigma-Aldrich, St. Louis, Missouri, USA), P-AKT(S473) (1:1000, 4060, Cell Signaling), P-PAK1(S144) (1:1000, 2606, Cell Signaling), p27 (1:1000, 3656, Cell Signaling), GAPDH (1:10000, sc-32233, Santa Cruz, Dallas, Texas, USA), HDAC1 (1:1000, sc-81598, Santa Cruz), and CXCL10 (1:1000, 14969, Cell Signaling).

Techniques: Migration, Western Blot, Control, One-tailed Test

Journal: Journal of Bone Oncology

Article Title: The metastatic role of the CXCL10-CXCR3 axis and its therapeutic potential in osteosarcoma

doi: 10.1016/j.jbo.2025.100690

Figure Lengend Snippet: Phenotypic analyses of the orthotopic xenograft mouse model of CXCR3 deletion. A, Sequence alignment confirms CRISPR-mediated CXCR3 deletion (KO). The top sequence (WT) corresponds to the reference genomic sequence of CXCR3, while the bottom sequence (KO) represents the mutant CXCR3 sequence in the KO cells. Red and magenta-colored nucleotides are the respective sgRNA1 and sgRNA2 used in the CRISPR editing. B, CXCR3 immunoblotting of parental 143B-Luc (WT) and KO cells. C, CXCL10-mediated transwell migration assays of parental WT and KO cells in 0.1 % FBS DMEM, n = 3. D, CCK8 proliferation assays of WT and KO cells in 0.1 % FBS DMEM. E, Bioluminescence images of orthotopic xenograft mouse models taken 5 weeks after intratibial injection with WT (left panel) and KO cells (right panel). F, Comparisons of volumes, calculated as 0.5 x h x w 2 (left), and bioluminescence (right) of primary tumors for the mouse models. G, Representative H&E staining images (left) and quantification (right) of the number and the area of metastatic nodules in resected mouse lungs, with each dot denoting one mouse. Error bars and asterisks represent standard deviations and statistical significance; n = three independent experiments; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns = not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were: CXCR3 (1:1000, PA5-28741, Invitrogen), Vinculin (1:5000, V9131, Sigma-Aldrich, St. Louis, Missouri, USA), P-AKT(S473) (1:1000, 4060, Cell Signaling), P-PAK1(S144) (1:1000, 2606, Cell Signaling), p27 (1:1000, 3656, Cell Signaling), GAPDH (1:10000, sc-32233, Santa Cruz, Dallas, Texas, USA), HDAC1 (1:1000, sc-81598, Santa Cruz), and CXCL10 (1:1000, 14969, Cell Signaling).

Techniques: Sequencing, CRISPR, Mutagenesis, Western Blot, Migration, Injection, Staining

Journal: Journal of Bone Oncology

Article Title: The metastatic role of the CXCL10-CXCR3 axis and its therapeutic potential in osteosarcoma

doi: 10.1016/j.jbo.2025.100690

Figure Lengend Snippet: The effects of CXCR3 isoforms in the CXCR3 deletion mutant. A, PCR analysis of the CXCR3 allele in parental 143B-Luc (WT), CXCR3 deletion mutant (KO), and CXCR3A and CXCR3B transfectants. The presence of a 761 bp band and a 370 bp band indicates the WT genomic CXCR3 gene and KO/CXCR3A isoform, respectively. The presence of both 761 bp and 370 bp bands indicates the presence of the CXCR3B isoform. CXCR3A transfectant clone 4 and CXCR3B transfectant clone 11 were selected for in vitro and animal experiments. B1 represents CXCR3B transfectant clone 1. B, CXCR3 immunoblotting of WT, KO, and CXCR3A (A) and CXCR3B (B) transfectants. C, CXCL10-mediated transwell migration assays of CXCR3A and CXCR3B transfectants relative to the KO mutant in 0.1 % FBS-supplemented DMEM, n = 3. D, Bioluminescence images of orthotopic xenograft mouse models taken 5 weeks after intratibial injection with CXCR3A and CXCR3B transfectant cells. E, Comparisons of primary tumor volumes, calculated as 0.5 x h x w 2 (left panel), and bioluminescence (right panel) in orthotopic xenograft mouse models of CXCR3A and CXCR3B. F, Representative H&E images (left) and quantification (right) of the number and cross-sectional area of metastatic nodules in resected lungs from the CXCR3A and CXCR3B mouse models, with each dot denoting one mouse. Error bars and asterisks represent standard deviations and statistical significance; * P < 0.05; ** P < 0.01; ns = not significant.

Article Snippet: The primary antibodies used were: CXCR3 (1:1000, PA5-28741, Invitrogen), Vinculin (1:5000, V9131, Sigma-Aldrich, St. Louis, Missouri, USA), P-AKT(S473) (1:1000, 4060, Cell Signaling), P-PAK1(S144) (1:1000, 2606, Cell Signaling), p27 (1:1000, 3656, Cell Signaling), GAPDH (1:10000, sc-32233, Santa Cruz, Dallas, Texas, USA), HDAC1 (1:1000, sc-81598, Santa Cruz), and CXCL10 (1:1000, 14969, Cell Signaling).

Techniques: Mutagenesis, Transfection, In Vitro, Western Blot, Migration, Injection

Journal: Journal of Bone Oncology

Article Title: The metastatic role of the CXCL10-CXCR3 axis and its therapeutic potential in osteosarcoma

doi: 10.1016/j.jbo.2025.100690

Figure Lengend Snippet: Therapeutic implications of CXCR3 inhibition. A, CXCL10-mediated chemotaxis of OS cell lines in transwell migration assays with and without AMG487 (AMG), a potent CXCR3 inhibitor. Overnight starved cells in 0.1 % FBS-supplemented DMEM were treated with 100 ng/mL CXCL10 and 1 µg/mL AMG, with cells pre-incubated with AMG for 1 h before chemotaxis, n = 3. B, Bioluminescence images of orthotopic xenograft mouse models of 143B-Luc taken 5 weeks after treatment with vehicle (left panel) and AMG (right panel). C, Comparisons of primary tumor volumes, calculated as 0.5 x h x w 2 (left panel), and bioluminescence (right panel) of AMG- or vehicle-treated orthotopic xenograft mouse models of 143B-Luc. D, Representative H&E images (left) and quantification of the number and the area (right) of metastatic nodules in resected lungs from AMG- and vehicle-treated mouse models, with each dot denoting one mouse. Error bars and asterisks represent standard deviations and statistical significance; * P < 0.05; ** P < 0.01; ns = not significant.

Article Snippet: The primary antibodies used were: CXCR3 (1:1000, PA5-28741, Invitrogen), Vinculin (1:5000, V9131, Sigma-Aldrich, St. Louis, Missouri, USA), P-AKT(S473) (1:1000, 4060, Cell Signaling), P-PAK1(S144) (1:1000, 2606, Cell Signaling), p27 (1:1000, 3656, Cell Signaling), GAPDH (1:10000, sc-32233, Santa Cruz, Dallas, Texas, USA), HDAC1 (1:1000, sc-81598, Santa Cruz), and CXCL10 (1:1000, 14969, Cell Signaling).

Techniques: Inhibition, Chemotaxis Assay, Migration, Incubation

Journal: Journal of Bone Oncology

Article Title: The metastatic role of the CXCL10-CXCR3 axis and its therapeutic potential in osteosarcoma

doi: 10.1016/j.jbo.2025.100690

Figure Lengend Snippet: Effects of CXCL10-CXCR3 signaling on AKT and PAK1 phosphorylation. A, Phospho-AKT (P-AKT) immunoblotting of 3 OS cell lines incubated for 2 min or 120 min with various concentrations of CXCL10 in 0.1 % FBS-supplemented DMEM. B, Phospho-AKT (P-AKT) immunoblotting of WT and KO cells treated for 5 min or 120 min with 2 concentrations of CXCL10 in 0.1 % FBS-supplemented DMEM. C, P-PAK1 immunoblotting of 4 OS parental and mutant cell lines incubated with 10 and 100 ng/mL CXCL10 in 0.1 % FBS-supplemented DMEM. GAPDH was used as a loading control. Cells incubated with 10 % and 0.1 % FBS-supplemented medium only served as positive (FBS) and negative (CTL) controls, respectively.

Article Snippet: The primary antibodies used were: CXCR3 (1:1000, PA5-28741, Invitrogen), Vinculin (1:5000, V9131, Sigma-Aldrich, St. Louis, Missouri, USA), P-AKT(S473) (1:1000, 4060, Cell Signaling), P-PAK1(S144) (1:1000, 2606, Cell Signaling), p27 (1:1000, 3656, Cell Signaling), GAPDH (1:10000, sc-32233, Santa Cruz, Dallas, Texas, USA), HDAC1 (1:1000, sc-81598, Santa Cruz), and CXCL10 (1:1000, 14969, Cell Signaling).

Techniques: Phospho-proteomics, Western Blot, Incubation, Mutagenesis, Control