Journal: Life Science Alliance

Article Title: Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression

doi: 10.26508/lsa.202302041

Figure Lengend Snippet: (A) Box plots of cell viability (MTT) assays ± calcitonin gene–related peptide (CGRP) of human cancer cell lines (LNCaP, PC-3, DU145, MCF-7, ZR75-1, MDA MB-231, SK-MES-1, and A549) for 48 h. P ≤ 0.05 is considered as statistically significant (one-way ANOVA). (B, C, D, E) IncuCyte ZOOM cell proliferation assays ± CGRP of human bone metastatic cancer cell lines (B) PC-3, (C) DU145, (D) MDA MB-231, and (E) A549 for 72 h. Data are the mean ± SEM. Significance versus vehicle (mixed-effects models). (F) Doubling time ± CGRP of human bone metastatic cancer cell lines (PC-3, DU145, MDA MB-231, and A549) for 72 h. Data are the mean ± SEM. Significance versus vehicle ( t test). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. (G) Box plots of cell viability (MTT) assays ± calcitonin gene–related peptide (CGRP) ± CGRP 8-37 ( calcitonin receptor–like receptor inhibitor) of human cancer cell lines (PC3, DU145, MDA MB-231, and A549) for 48 h. P ≤ 0.05 is considered as statistically significant (one-way ANOVA, Tukey’s multiple comparisons). (H) IncuCyte ZOOM cell proliferation assays ± CGRP ± CGRP 8-37 of DU145 for 72 h. Data are the mean ± SEM. Significance versus vehicle (mixed-effects models). (I) Box plots of cell viability (MTT) assays ± substance P (SP) of human cancer cell lines (PC-3, DU145, ZR75-1, MDA MB-231, SK-MES-1, and A549) for 48 h. P ≤ 0.05 is considered as statistically significant (one-way ANOVA). (J) Plasma SP levels of prostate cancer patients without (n = 11) and with (n = 22) bone metastasis. Data are the mean ± SEM ( t test). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: In some cases, cells were pretreated with the CGRP receptor antagonist (1 nM, CGRP 8-37, Cat. #: 1181; Tocris Bioscience) or p38 inhibitor (5 μM, SB203580, Cat. #: S1076; Selleckchem) for 1 h before the CGRP treatment.

Techniques: Clinical Proteomics

Journal: Life Science Alliance

Article Title: Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression

doi: 10.26508/lsa.202302041

Figure Lengend Snippet: (A) MTT assay of RM-1 treated with 0–100 nM of murine CGRP. Data are the mean ± SEM. * P ≤ 0.05 versus vehicle (one-way ANOVA, Tukey’s multiple comparisons). (B) Representative images of antibody-based cell pathway array data presented in . (C) Representative Western blot of p38 and HSP27 phosphorylation ± CGRP. GAPDH was used as a loading control.

Article Snippet: In some cases, cells were pretreated with the CGRP receptor antagonist (1 nM, CGRP 8-37, Cat. #: 1181; Tocris Bioscience) or p38 inhibitor (5 μM, SB203580, Cat. #: S1076; Selleckchem) for 1 h before the CGRP treatment.

Techniques: MTT Assay, Western Blot, Phospho-proteomics, Control

Journal: Life Science Alliance

Article Title: Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression

doi: 10.26508/lsa.202302041

Figure Lengend Snippet: (A) Quantification of antibody-based cell pathway array data. DU145 and MDA-MB-231 cells (responders to CGRP), and LNCaP and MCF-7 cells (non-responders to CGRP) were exposed to CGRP for 60 min. (B) Representative Western blot of p38 and HSP27 phosphorylation ± CGRP ± SB203580 (p38 inhibitor) of human cancer cell lines (PC-3, DU145, MDA MB-231, and A549). GAPDH was used as a loading control. (C) Box plots of cell viability (MTT) assays ± CGRP ± SB203580 of human cancer cell lines (PC-3, DU145, MDA MB-231, and A549) for 48 h. P ≤ 0.05 is considered as statistically significant (one-way ANOVA, Tukey’s multiple comparisons). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: In some cases, cells were pretreated with the CGRP receptor antagonist (1 nM, CGRP 8-37, Cat. #: 1181; Tocris Bioscience) or p38 inhibitor (5 μM, SB203580, Cat. #: S1076; Selleckchem) for 1 h before the CGRP treatment.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Life Science Alliance

Article Title: Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression

doi: 10.26508/lsa.202302041

Figure Lengend Snippet: (A) Representative Western blot of p38 and HSP27 phosphorylation ± CGRP ± CGRP8-37 ( CGRP receptor antagonist ) of human cancer cell lines (PC-3, DU145, MDA MB-231, and A549). GAPDH was used as a loading control. (B) Experimental schedule. Luciferase-expressing murine prostate cancer cell line RM-1 was implanted directly into femurs of C57BL/6 WT mice (n = 10/group). These mice were treated daily with either vehicle or CGRP8-37. (C) Bone remodeling was measured by X-ray. Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant (mixed-effects model). (D) Pain behavior was measured by guarding behavior measurement. Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant ( t test). (E) Bone metastatic growth was measured by bioluminescence imaging. Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant (mixed-effects model). (B, C, D, E, F) Representative images of total and phosphorylated p38–immunostained bone marrow of animals in (B, C, D, E). DAPI is used for nuclear staining. ×10. Bar = 100 μm. (F, G) Quantification of (F). Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant ( t test).

Article Snippet: In some cases, cells were pretreated with the CGRP receptor antagonist (1 nM, CGRP 8-37, Cat. #: 1181; Tocris Bioscience) or p38 inhibitor (5 μM, SB203580, Cat. #: S1076; Selleckchem) for 1 h before the CGRP treatment.

Techniques: Western Blot, Phospho-proteomics, Control, Luciferase, Expressing, Imaging, Staining

Journal: Life Science Alliance

Article Title: Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression

doi: 10.26508/lsa.202302041

Figure Lengend Snippet: (A) Experimental schedule. Luciferase-expressing murine prostate cancer cell line RM-1 was implanted directly into femurs of C57BL/6 WT mice (n = 20/group). These mice were treated intraperitoneally with either isotype control antibody (Ab) or anti-CGRP monoclonal Ab (30 mg/kg) at day 6, 13, or 20. (B) Bone remodeling was measured by X-ray. Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant ( t test). (C) Pain behavior was measured by guarding behavior measurement. Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant ( t test). (D) Bone metastatic growth was measured by bioluminescence imaging. Data are the mean ± SEM. * P ≤ 0.05 versus isotype control antibody (Ab) (mixed-effects model). (A, B, C, D, E) Representative images of TRAP-positive osteoclasts in the bone marrow of animals in (A, B, C, D). ×20. Bar = 100 μm. (F) Quantification of (E). Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant ( t test). (A, B, C, D, G) Representative images of total and phosphorylated p38–immunostained bone marrow of animals in (A, B, C, D). DAPI is used for nuclear staining. ×10. Bar = 100 μm. (G, H) Quantification of (G). Data are the mean ± SEM. P ≤ 0.05 is considered as statistically significant ( t test).

Article Snippet: In some cases, cells were pretreated with the CGRP receptor antagonist (1 nM, CGRP 8-37, Cat. #: 1181; Tocris Bioscience) or p38 inhibitor (5 μM, SB203580, Cat. #: S1076; Selleckchem) for 1 h before the CGRP treatment.

Techniques: Luciferase, Expressing, Control, Imaging, Staining