Journal: International Journal of Molecular Sciences

Article Title: A ‘Spicy’ Mechanotransduction Switch: Capsaicin-Activated TRPV1 Receptor Modulates Osteosarcoma Cell Behavior and Drug Sensitivity

doi: 10.3390/ijms26188816

Figure Lengend Snippet: 1 Hz–24 h mechanical stretch induces cell orientation in U-2 OS cells via TRPV1 activation. ( A ) Representative fluorescence microscopy images of U-2 OS cells stained with Hoechst (nuclei), β-tubulin (cytoskeleton), and merged channels. Images were acquired at 40× magnification. Scale bar = 25 μm. ( B ) Schematic representation of nuclear orientation expressed as ⟨cos 2 φ⟩. ( C ) Violin plots display the distribution of nuclear orientation values in SAOS-2, U-2 OS, and hFOB cells. ( D ) Violin plots show nuclear orientation in stretched U-2 OS cells treated with AMG (I), Cytochalasin D (Cyto D) or Indomethacin (Indo). All analyses were performed on three biological replicates, with at least 45 cells analyzed per condition.

Article Snippet: Osteosarcoma cell lines Human SAOS-2 (HTB-85) and U-2 OS (HTB-96) and human fetal osteoblast cell line hFOB (CRL-3602) were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activation Assay, Fluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: A ‘Spicy’ Mechanotransduction Switch: Capsaicin-Activated TRPV1 Receptor Modulates Osteosarcoma Cell Behavior and Drug Sensitivity

doi: 10.3390/ijms26188816

Figure Lengend Snippet: Capsaicin and its Antagonist AMG9810 Modulate OS Mechanotransduction by Activating or Blocking TRPV1 Channels. ( A ) Representative fluorescence microscopy images showing the effects of ASP7633 (ASP) and Capsaicin (CAP) with or without AMG (CAP+ I) on nuclear morphology. Magnification: 20×; scale bar: 25 µm. Nuclear enlargement quantification is shown as bar plots. Data represent three biological replicates, with a minimum of 30 cells per condition. ( B ) Representative confocal microscopy images of OS cells, with corresponding bar plot quantification of the nucleus-to-cytoplasm (N/C) ratio following the same treatments as in ( A ). Magnification: 20×, scale bar: 100 μm. The analysis includes three biological replicates with at least 25 cells per condition. ( C ) Migration assays showing the impact of capsaicin, with or without AMG9810, on the migratory ability of both OS cell lines. ( D ) The effect of Capsaicin treatment (with or without co-treatment with AMG9810) on the adhesion capability of U-2 OS and SAOS-2 cells. Scale bar: 100 μm. ( E ) Shows the relative cell viability of hFOB, U-2 OS, and SAOS-2 cell lines after treatment with increasing concentrations of capsaicin. ( F ) Relative cytotoxicity of U-2 OS cells treated with cisplatin or doxorubicin following capsaicin treatment with or without AMG. ( G ) Relative cytotoxicity of SAOS-2 cells treated with cisplatin or doxorubicin following capsaicin treatment with or without AMG. For each experiment, statistical analyses were performed on the most representative of three biological replicates, each comprising a minimum of four technical replicates per condition. Statistical significance between treated and control samples was determined using Student’s t -test, with significance levels indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Article Snippet: Osteosarcoma cell lines Human SAOS-2 (HTB-85) and U-2 OS (HTB-96) and human fetal osteoblast cell line hFOB (CRL-3602) were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Blocking Assay, Fluorescence, Microscopy, Confocal Microscopy, Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: A ‘Spicy’ Mechanotransduction Switch: Capsaicin-Activated TRPV1 Receptor Modulates Osteosarcoma Cell Behavior and Drug Sensitivity

doi: 10.3390/ijms26188816

Figure Lengend Snippet: TRPV1 activation modulates expression and nuclear localization Src in OS cells. ( A – D ) Representative Western blot images and their respective densitometric analyses of cell extract from U-2 OS or SAOS-2. ( A , C ) SRC (60 kDa) and GAPDH (37 kDa) protein levels following mechanical stretch or capsaicin treatment, with or without co-treatment with AMG9810, respectively. ( B , D ) Acetylated histone 3 (acH3, 17 kDa) and Histone 3 (H3 17 kDa) levels under the same conditions as above. ( E , F ) Representative immunofluorescence images of nuclei (Hoechst), Src protein, and merged channels in OS cells subjected to mechanical stretch or capsaicin treatment, with or without co-treatment with AMG9810. Src nuclear levels quantified by corrected total cell fluorescence (CTCF) are reported as bar plots (mean ± SD). ( G , H ) Western blot and CTCF analysis of Src in hFOB cells under mechanical stretch or capsaicin treatment. Densitometric analysis of the Western blot bands was performed using ImageJ 1.52 software and quantified in arbitrary units. CellProfiler 4.2.8 was used to identify nuclear SRC fluorescence intensity and area values . Data represent three biological replicates, with a minimum of 45 cells analyzed per condition. Error bars represent the standard error of the mean (SEM). Statistical analysis was conducted on at least three biological replicates. Statistical significance was determined using an unpaired t -test, with results shown as mean ± SD; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Article Snippet: Osteosarcoma cell lines Human SAOS-2 (HTB-85) and U-2 OS (HTB-96) and human fetal osteoblast cell line hFOB (CRL-3602) were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software