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

Journal: The Journal of cell biology

Article Title: Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

doi: 10.1083/jcb.202305048

Figure Lengend Snippet: Figure 1. Transcriptional reprogramming of neuronal metabolism during glucose deprivation. (A) Schematic for glucose deprivation of rat cortical neurons. A subset of ∼250 genes significantly upregulated during glucose deprivation (P value <0.01) were selected for pathway analysis using annotated gene sets from the Molecular Signatures Database (MSigDB). n = 3 cortical samples from one rat litter. (B) Enrichment of genes in MSigDB Reactome pathways with adjusted P value <0.05. (C) Enrichment of the target genes of the CREB transcription factor in glucose-deprived neurons (adjusted P value <0.05). The target gene Pgc1α (PPARGC1A) is highlighted in red. (D) Immunostaining of cortical neuronal cultures (treated as in A) with an anti-phospho-CREB antibody and Hoechst nuclear stain. Arrowheads denote pCREB-positive nuclei. (E) Fraction of nuclei with positive p-CREB staining in fields of view (FOV), determined as described in Materials and methods. % total ± SEM: +glucose (3 h), 14.87 ± 2.29, −glucose (3 h), 27.41 ± 3.3. n = 17–22 FOVs. (F) Relative mRNA expression of Pgc1α in neuronal cultures treated as in A, with or without the AMPK inhibitor, dorsomorphin. Values are normalized to β-actin mRNA and expressed relative to the +glucose condition. Average normalized mRNA level ± SEM: −glucose (1 h), 2.83 ± 0.42; −glucose (3 h), 3.67 ± 0.46; −glucose + dorsomorphin (3 h), 1.32 ± 0.22. n = 3–10 cortical samples. Bar graphs are plotted as mean ± SEM. Mann–Whitney U test (E), one-way ANOVA (F). See Table 1.

Article Snippet: The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/ HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat).

Techniques: Immunostaining, Staining, Expressing, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

doi: 10.1083/jcb.202305048

Figure Lengend Snippet: Figure 2. Glucose deprivation stimulates neuronal Sirt3 expression and deacetylation of mitochondrial proteins. (A) Relative Sirt3 mRNA expression in control and glucose-deprived neu- rons. Values are normalized to β-actin mRNA and expressed relative to control (+glucose). n = 3–10 cortical samples. Average normalized mRNA level ± SEM: −glucose (1 h), 2.58 ± 0.62; −glucose (3 h), 2.20 ± 0.27; −glucose + dorsomorphin (3 h), 1.12 ± 0.08. n = 3–11 cortical samples. (B) Relative Sirt3 mRNA expression in cultures transduced with adenoviral particles encoding GFP (control) and GFP-PGC1α, normalized to 18s rRNA and ex- pressed relative to control. Average normalized mRNA level ± SEM: GFP-PGC1α, 1.93 ± 0.30. n = 16 cortical samples/condition. (C) Immunoblotting of Sirt3 protein expression in cortical neurons with antibodies against Sirt3 and the cytosolic and mitochondrial controls, β-actin, and ATPase5β, respectively. (D) Sirt3 band intensity normalized to the ATPase5β and expressed relative to control. Average normalized Sirt3 band intensity ± SEM: +glucose, 0.99 ± 0.05; −glucose, 1.74 ± 0.16. n = 7 cortical samples/condition. (E) A paradigm for the analysis of Sirt3 expression in hippocampi of mice fed ad libitum (ad lib) or alternate-day fasted for 6 mo (ADF). Schematic created with https:// biorender.com. (F) Immunoblotting of Sirt3 protein in mouse hippocampal lysates. (G) Sirt3 band intensity normalized to the ATPase5β band and expressed relative to the ad lib mice. Average normalized Sirt3 band intensity ± SEM: ad lib, 1 ± 0.24; ADF (6 mo), 1.88 ± 0.19. n = 6 mice/condition. (H) Mitochondrial and cytosolic fractions isolated from cortical neuronal cul- tures maintained for 3 h with (+) or without (−) glucose. High and low exposures were 60 and 25 s, respectively. (I) Intensity of lysine acetylation bands normalized to α-tubulin or ATPase5β plot- ted relative to control. Average normalized Ac-K intensity: cytosolic fraction (−glucose), 1.21 ± 0.15; mitochondrial fraction (−glucose), 0.66 ± 0.08. n = 5 cortical samples. One-way ANOVA (A), two- tailed, unpaired t test (B), Mann–Whitney U test (D and G), one sample t test (I). See Table 1. Source data are available for this figure: SourceData F2.

Article Snippet: The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/ HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat).

Techniques: Expressing, Control, Transduction, Western Blot, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Biochemistry

Article Title: The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP

doi: 10.1021/acs.biochem.7b00117

Figure Lengend Snippet: Western blot and immunofluorescence analysis of PKA. (A) Western blot for RIIβ showing antibody specificity. Lanes are 1000-, 2000-, and 5000-fold dilutions for RIIβ (lanes 1–3, respectively), RIα, − and RIIα. − (B) Western blot showing the presence of RIIβ in FDB, EDB, and soleus muscle tissue. (C) Representative confocal images of a segment of FDB fiber indirectly immunolabeled with antibodies against RIIβ (red), α-actinin (cyan), dystrophin (green), and POPO-1, to define the nuclei. (D) Close-ups (left) of the boxed region indicated in panel C for RIIβ (top), α-actinin (middle), and merged images (bottom) and averaged fluorescence profiles (right) of RIIβ (red trace) and α-actinin (blue trace) signals across the box. (E and F) Same labeling as in panels C and D, respectively, except that anti-RIIβ was not included. Scale bars in panels C and E are 20 μm and in panels D and F are 2 μm.

Article Snippet: All antibodies used are commercially available as follows: RIIβ (1:100; ab75993), α-actinin (1:250; A7811, Sigma, St. Louis, MO), dystrophin (1:100; MANDRA1, Developmental Studies Hybridoma Bank, Iowa City, IA), Alexa-488 goat anti-mouse, Alexa-568 goat anti-mouse, and Alexa-647 goat anti-rabbit (1:1000; A21244, A11004, and A11029, respectively, Thermo-Fisher, Rockford, IL).

Techniques: Western Blot, Immunofluorescence, Immunolabeling, Fluorescence, Labeling

Journal: Cells

Article Title: VDR Regulates BNP Promoting Neurite Growth and Survival of Cochlear Spiral Ganglion Neurons through cGMP-PKG Signaling Pathway

doi: 10.3390/cells11233746

Figure Lengend Snippet: Vitamin D receptor (VDR) expression in rat spiral ganglion neurons and association between VDR overexpression and BNP expression. ( A ) VDR is expressed in rat spiral ganglion neurons and remains unchanged during the whole development, overlapping with BNP. ( B ) PCR analysis of BNP expression after VDR overexpression. Cochlear explants transfected with negative control (NC) lentiviral served as controls. Overexpression of VDR increased BNP RNA levels. ( C ) β-III Tubulin positive spiral ganglion neurons of cochlear explants were infected with GFP-tagged VDR overexpression lentivirus. ( D ) Western blot analysis of BNP at 48 h after transfection in rat cochlear explants and 293 T cells, using GAPDH as the endogenous housekeeping control gene. Cochlear explants transfected with negative control lentiviral served as controls. ( E , F ) Histograms for Western blot results show the level of BNP tended to increase with an increased level of VDR in rat cochlear and 293 T cells. All of the data are presented as the mean ± SD of three independent experiments. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001compared with the NC.

Article Snippet: The antibodies used in immunofluorescence included polyclonal rabbit anti-BNP antibody (1: 400; PA5-96084, Thermo Fisher Scientific, Waltham, MA, USA), polyclonal rabbit anti-NPRA antibody (1: 500; PA5-29049, Thermo Fisher Scientific, Waltham, MA, USA), monoclonal mouse anti-tubulin β-III primary antibody (1:400; ab78078, Abcam, Cambridge, UK), polyclonal chicken anti-tubulin β-III antibody (1:500; GTX85469, GeneTex, Irvine, TX, USA), Alexa Fluor 488 conjugated donkey anti-mouse IgG (1: 400; A-21202, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1: 400; A-21207, Thermo Fisher Scientific, Waltham, MA, USA), and Alexa Fluor 647-conjugated goat anti-chicken IgY (1:400; ab150176, Abcam, Cambridge, UK).

Techniques: Expressing, Over Expression, Transfection, Negative Control, Infection, Western Blot, Control