Journal: Smart Materials in Medicine

Article Title: Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

doi: 10.1016/j.smaim.2023.04.001

Figure Lengend Snippet: Fig. 10. Confocal microscopic inspection of OPN and collagen I expression of MC-3T3-E1 cells in 3D spheroids with ACP particles embedded and 2D culture system, respectively. (A) Left panel: OPN and collagen I expression in different culture media in 3D reconstructed images; Right panel: maximal fluorescent projection of OPN and collagen I in 3D spheroids as shown in left penal; (B) OPN and collagen I expression in different culture media in a 2D culture system. Scale bar ¼ 100 μm.

Article Snippet: In brief, cells and spheroids were cultured for 7 days and then fixed with 4 wt % PFA for 20 min. After washing in PBS thrice, samples were treated with 1% (v/v) TritionX-100 (X100, Sigma-Aldrich, USA) for 20 min, followed by blocking in 1 wt % BSA/PBS solution for 1 h. Then, the samples were incubated in goat-anti-mouse OPN primary antibody (Ab) (AF808, R&D systems, USA) and rabbit-anti-mouse collagen I Ab (ab21286, Abcam, USA) at 4 C overnight.

Techniques: Expressing

Journal: Journal of oral biosciences

Article Title: Disruption of CADM1-dependent cell-cell adhesion in human oral squamous cell carcinoma cells results in tumor progression, possibly through an increase of MMP-2 and MMP-9 expression.

doi: 10.1016/j.job.2023.11.005

Figure Lengend Snippet: Fig. 1. Investigation of the expression levels of EMT-related molecules in hOSCC HSC-3 and LMF4 cells. A)~D) The mRNA expression levels of epithelial markers, A) E-cadherin and C) cytokeratin 18, and mesenchymal markers, B) N-cadherin and D) vimentin in HSC-3 cells (gray bar) and LMF4 cells (black bar) were analyzed using RT-qPCR. Values were normalized to GAPDH mRNA levels. Data are presented as the mean ± SD of quadruplicate experiments. Differences in values between HSC-3 and LMF4 cells were statistically analyzed using Student’s t-test (**P < 0.01 and *P < 0.05). E) The protein expression levels of E-cadherin and N-cadherin were analyzed using western blot analysis. Western blot analysis was repeated three times, and the repre sentative data were indicated. For the statistical evaluation of the obtained band intensity, β-actin was used as the loading standard, and the values obtained from the concentration of each band were normalized to β-actin protein levels. Data are presented as the mean ± SD of triplicate experiments. Differences in values between HSC-3 and LMF4 cells were statistically analyzed using Student’s t-test (**P < 0.01 and *P < 0.05).

Article Snippet: The target proteins were analyzed using chicken anti-SynCAM mAb primary antibody (at a 1:200 dilution, clone 3E1; CM004-3, Medical and Biological Laboratories (MBL), Tokyo, Japan), while a mouse anti-β-actin antibody (at a 1:1000 dilution, clone C4; Santa Cruz Biotechnology) was used as the loading control on the polyvinylidene fluoride membranes (Merck Millipore).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay

Journal: Journal of oral biosciences

Article Title: Disruption of CADM1-dependent cell-cell adhesion in human oral squamous cell carcinoma cells results in tumor progression, possibly through an increase of MMP-2 and MMP-9 expression.

doi: 10.1016/j.job.2023.11.005

Figure Lengend Snippet: Fig. 3. Investigation of the expression level of tumor-related cell adhesion molecule CADM1 in HSC-3 and LMF4 cells. A) The level of CADM1 mRNA in HSC-3 cells (gray bar) and LMF4 cells (black bar) was examined using RT-qPCR. Data are presented as the mean ± SD of quadruplicate experiments. B) The protein expression levels of CADM1 were analyzed using Western blot analysis. Western blot analysis was repeated three times, and the representative data were indicated. For the statistical evaluation of the obtained band intensity, β-actin was used as the loading standard, and the values obtained from the concentration of each band were normalized to β-actin protein levels. Differences in values between HSC-3 and LMF4 cells were statistically analyzed using Student’s t-test (**P < 0.01 and *P < 0.05). C) The localization and intensity of CADM1 in HSC-3 and LMF4 cells were examined by fluorescence immunostaining. Both cells were immunostained with anti-CADM1 antibody (green), and with phalloidin (red) to detect F-actin and DAPI (blue) to detect nuclei. Scale bars represent 25 μm.

Article Snippet: The target proteins were analyzed using chicken anti-SynCAM mAb primary antibody (at a 1:200 dilution, clone 3E1; CM004-3, Medical and Biological Laboratories (MBL), Tokyo, Japan), while a mouse anti-β-actin antibody (at a 1:1000 dilution, clone C4; Santa Cruz Biotechnology) was used as the loading control on the polyvinylidene fluoride membranes (Merck Millipore).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Fluorescence, Immunostaining

Journal: Journal of oral biosciences

Article Title: Disruption of CADM1-dependent cell-cell adhesion in human oral squamous cell carcinoma cells results in tumor progression, possibly through an increase of MMP-2 and MMP-9 expression.

doi: 10.1016/j.job.2023.11.005

Figure Lengend Snippet: Fig. 4. Downregulation of CADM1 expression level with si-CADM1 increased expression of MMP-2 and MMP-9 in HSC-3 cells. A) The mRNA expression levels of CADM1 in HSC-3 cells treated with si-control or si-CADM1 were analyzed using RT-qPCR. B) The expression levels of CADM1 in HSC-3 cells treated with si-control or si-CADM1 were analyzed using western blotting. Western blot analysis was repeated three times, and the representative data were indicated. For the statistical evaluation of the obtained band intensity, β-actin was used as the loading standard, and the values obtained from the intensity of each band were normalized to β-actin protein levels. Differences in values between si-control and si-CADM1 in HSC-3 cells were statistically analyzed using Student’s t-test (*P < 0.05). C) The comparison of expression levels of MMP-2 between si-control-treated-, and si-CADM1-treated-HSC-3 cells (left graph) and that between HSC-3 and LMF4 cells (right graph) were analyzed using RT-qPCR. D) The comparison of expression levels of MMP-9 between si-control-treated- and si-CADM1- treated-HSC-3 cells (left graph) and that between HSC-3 and LMF4 cells (right graph) were analyzed using RT-qPCR. Values were normalized to GAPDH mRNA levels. Data are presented as the mean ± SD of quadruplicate experiments. Differences in the values between si-control-treated- and si-CADM1-treated-HSC-3 cells or that between HSC-3 and LMF4 cells were statistically analyzed using Student’s t-test (**P < 0.01 and *P < 0.05).

Article Snippet: The target proteins were analyzed using chicken anti-SynCAM mAb primary antibody (at a 1:200 dilution, clone 3E1; CM004-3, Medical and Biological Laboratories (MBL), Tokyo, Japan), while a mouse anti-β-actin antibody (at a 1:1000 dilution, clone C4; Santa Cruz Biotechnology) was used as the loading control on the polyvinylidene fluoride membranes (Merck Millipore).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Comparison