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: Human molecular genetics

Article Title: Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy.

doi: 10.1093/hmg/ddu451

Figure Lengend Snippet: Figure 4. P0-Cre-mediated recombination efficiency in the Fig4Floxed/plt, P0-Cre model. (A) PCR analysis on genomic DNA from Fig4Floxed/plt, P0-Cre mice and controls. A 350-bp recombination band was detected in the nerve of mutants but not in wild type. The 350-bp faint band in the brain and spinal cord indicates recom- binationin cranial and spinal nerves of brain and spinal cord, respectively. (B) Westernblot analysisof nerve homogenates at P30 indicates decreased Fig4 expression in sciatic nerves of Fig4Floxed/plt, P0-Cre mice. (C–F) Purified Schwann cells from Fig4Floxed/plt, P0-Cre mouse nerves at P3 indicate the presence of enlarged LE/LY-LAMP1 positive (green in C, C′, E, E′; red is phalloidin; blue is DAPI) in mutant cells, a feature of Fig4 loss and PtdIns(3,5)P2 decrease. (D) S100 staining marks Schwann cells in the culture. Bar in (D) is 20 mm for (C, C′, E, E′) and 50 mm for (D).

Article Snippet: For western blot analysis and immunohistochemistry the following antibodies were used: goat anti-ChAT (Millipore); rat anti-LAMP1 (Iowa Hybridoma bank); Guinea pig anti-P62 (Progen); rabbit anti-LC3 (Sigma); rat anti-MBP (Chemicon); rabbit anti-NF-H (Millipore); mouse anti-TUJ1 (Promega); mouse anti-tubulin (Sigma); rabbit anti-actin (Sigma); mouse anti-FIG4 (Neuromab); rabbit anti-S100 (Sigma), rabbit anti-NGF receptor p75 (Millipore); chicken anti-P0 (Millipore) and mouse anti-P0 antibodies (kindly provided by J.J. Archelos); rabbit anti-ubiquitin (Santa Cruz); rhodamine phalloidin (Molecular Probes).

Techniques: Expressing, Mutagenesis, Staining

Journal: Human molecular genetics

Article Title: Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy.

doi: 10.1093/hmg/ddu451

Figure Lengend Snippet: Figure 5. Nerve regeneration is impaired in the Fig4Floxed/plt, P0-Cre conditional knock out. (A) Semithin section analysis of sciatic nerves from controls (left) and Fig4Floxed/plt, P0-Cre mutant nerves (right panels) analysed at 60 dpi (days post injury-crush performed at 2.5 months) indicates that regeneration is impaired in this mutant. Fibre loss is also observed in mutant nerves after injury. (B) The distribution of the number of axons per diameter indicates that fibres in the range of 3–5 mm (P ¼ 0.006) and 5–7 mm of diameter (P ¼ 0.001) were significantly decreased in the Fig4Floxed/plt, P0-Cre nerves after crush. (C) Staining of Fig4Floxed/plt, P0-Cre nerves at 7 dpi using anti-MBP antibody (green) which marks myelin and anti-p75 (red), which labels trans-differentiating Schwann cells. Bar in (A) is 20 mm; in (C) is 100 mm.

Article Snippet: For western blot analysis and immunohistochemistry the following antibodies were used: goat anti-ChAT (Millipore); rat anti-LAMP1 (Iowa Hybridoma bank); Guinea pig anti-P62 (Progen); rabbit anti-LC3 (Sigma); rat anti-MBP (Chemicon); rabbit anti-NF-H (Millipore); mouse anti-TUJ1 (Promega); mouse anti-tubulin (Sigma); rabbit anti-actin (Sigma); mouse anti-FIG4 (Neuromab); rabbit anti-S100 (Sigma), rabbit anti-NGF receptor p75 (Millipore); chicken anti-P0 (Millipore) and mouse anti-P0 antibodies (kindly provided by J.J. Archelos); rabbit anti-ubiquitin (Santa Cruz); rhodamine phalloidin (Molecular Probes).

Techniques: Knock-Out, Mutagenesis, Staining

Journal: Human molecular genetics

Article Title: Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy.

doi: 10.1093/hmg/ddu451

Figure Lengend Snippet: Figure 6. Schwann cell/DRG neuron co-cultures from Fig4Floxed/plt, P0-Cre mice are hypomyelinated. (A and B) Bright field images showing vacuolization of Schwann cells in explants established from Fig4Floxed/plt, P0-Cre mouse embryos, which corresponds to LE/LY compartment LAMP1 positive (LAMP1 green, red phalloidin and blue DAPI) in (C and D). (E–H) Vacuolated Schwann cells produced less myelin segments after 7 days of ascorbic acid treatment when compared withcontrolcultures(redisMBP,greenisneurofilamentandblueisDAPI),n ¼ 6–8explantspergenotype/threedifferentexperiments. (IandJ)Myelinproteinzero, P0,doesnotco-localizewithLAMP1incontrolandinFig4Floxed/plt,P0-CreSchwanncells(greenisP0,redisLAMP1andblueisDAPI),n ¼ 4explantspertimepoint of ascorbic acid treatment, per genotype. Two different anti-P0 antibodies were used. Bar in (F) is 150 mm for (A and B) and 50 mm for (C–F); bar in (K) is 13 mm for (I–K).

Article Snippet: For western blot analysis and immunohistochemistry the following antibodies were used: goat anti-ChAT (Millipore); rat anti-LAMP1 (Iowa Hybridoma bank); Guinea pig anti-P62 (Progen); rabbit anti-LC3 (Sigma); rat anti-MBP (Chemicon); rabbit anti-NF-H (Millipore); mouse anti-TUJ1 (Promega); mouse anti-tubulin (Sigma); rabbit anti-actin (Sigma); mouse anti-FIG4 (Neuromab); rabbit anti-S100 (Sigma), rabbit anti-NGF receptor p75 (Millipore); chicken anti-P0 (Millipore) and mouse anti-P0 antibodies (kindly provided by J.J. Archelos); rabbit anti-ubiquitin (Santa Cruz); rhodamine phalloidin (Molecular Probes).

Techniques: Produced

Journal: Human molecular genetics

Article Title: Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy.

doi: 10.1093/hmg/ddu451

Figure Lengend Snippet: Figure8. Block of autophagicprogressionin Fig4Floxed/plt, P0-Cre nerves.(A–D) LAMP1,LC3II/I and p62 levels are increased in P30 sciatic nerves of Fig4Floxed/plt, P0-Cre mice. An elevation of p62 indicates a block in autophagic progression in Schwann cells. In (B), lysates from HeLa cells starved and starved-treated using Bafalomycin, which blocks lysosomal-mediated degradation, were used as controls of LC3 II/I elevation. In (C), brain lysates from Fig4plt/plt and controls were used as a positive control of p62 increase, as already reported (1,33). (E) Lysates of sciatic nerves from Fig4Floxed/plt, P0-Cre mice and controls at P90 were stained using an anti-polyubiquitin antibody. Control genotypes are Fig4Floxed/plt and Fig4Floxed/Floxed.

Article Snippet: For western blot analysis and immunohistochemistry the following antibodies were used: goat anti-ChAT (Millipore); rat anti-LAMP1 (Iowa Hybridoma bank); Guinea pig anti-P62 (Progen); rabbit anti-LC3 (Sigma); rat anti-MBP (Chemicon); rabbit anti-NF-H (Millipore); mouse anti-TUJ1 (Promega); mouse anti-tubulin (Sigma); rabbit anti-actin (Sigma); mouse anti-FIG4 (Neuromab); rabbit anti-S100 (Sigma), rabbit anti-NGF receptor p75 (Millipore); chicken anti-P0 (Millipore) and mouse anti-P0 antibodies (kindly provided by J.J. Archelos); rabbit anti-ubiquitin (Santa Cruz); rhodamine phalloidin (Molecular Probes).

Techniques: Blocking Assay, Positive Control, Staining, Control

Journal: Aging Cell

Article Title: G6PD overexpression protects from oxidative stress and age‐related hearing loss

doi: 10.1111/acel.13275

Figure Lengend Snippet: G6PD ‐Tg mice show better hearing and preserved cochlear cytoarchitecture. (A) Relative levels of total cochlear G6PD mRNA in G6PD ‐Tg and WT mice. Expression levels were calculated as the addition of the 2 −ΔCt for the murine and human G6PD primers using 18s as a reference gene and normalization to levels in 3‐month‐old WT mice. RT‐qPCR data are presented as mean ± SEM of triplicate measurements from pooled samples of three 3‐ and 9‐month‐old mice per condition, and mean ± SEM of 3 individual samples of 12‐month‐old mice. (B) G6PD and PGD activities measured from pooled samples of three cochleae in 3‐ and 9‐month‐old mice per condition. NADPH levels measured from pooled inner ears of three 3‐ and 9‐month‐old mice per condition. Data are presented relative to milligram of protein in samples. Data presented as mean ± SEM of at least duplicate measurements. Statistical significance was analyzed by Student's t test: * G6PD ‐Tg vs WT, # 9‐ and 12‐month‐old mice vs 3‐ and 9‐month‐old mice, respectively (*, # p < 0.05; **, ## p < 0.01; ***, ### p < 0.001). (C) ABR thresholds in response to click and tone burst stimuli (4, 8, 16, 28, 40 kHz) in 3‐, 6‐, 9‐, and 12‐month‐old WT and G6PD ‐Tg mice. (D) Representative ABR recordings in response to click stimulus of WT (left) and G6PD ‐Tg (right) mice at 9 months of age; thresholds are highlighted. Overlapped ABR waves recorded in response to 70 dB SPL stimuli in WT (black) and G6PD ‐Tg (green) mice. (E) Input–output ABR wave I amplitude and latency plotted against intensity (dB SPL) for 9‐month‐old mice. Statistical significance was analyzed by Student's t test: * G6PD ‐Tg vs WT in C and E (* p < 0.05; ** p < 0.01; *** p < 0.001). (F) Representative basal and middle turns hematoxylin–eosin staining and MyeP0 immunolabeling of paraffin‐embedded cochlear midmodiolar sections of 9‐month‐old G6PD ‐Tg and WT mice ( n = 4 each group). Scale bars: 125 µm in a, 50 µm in b, and c and 25 µm in d. (G) Cochlear gene expression of Prestin in 9‐month‐old mice. Expression levels were calculated as ΔRQ normalized to expression data for 3‐month‐old mice for each condition and using 18s as a reference gene. Values are presented as mean ± SEM of triplicate measurements from pooled samples of 3 mice per condition. (H) MyeP0 intensity of spiral ganglion neurons in 9‐month‐old mice; at least 3 different measurements per turn of each mouse were taken ( n = 4 mice per group). Values are presented as mean ± SEM. Statistical significance between genotypes was analyzed by Student's t test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Paraffin cochlear midmodiolar sections (5 μm) were obtained on a RM2155 microtome (Leica Microsystems, Deerfield, IL, USA) and were either hematoxylin–eosin‐stained or immunostained with chicken anti‐myelin P0 (MyeP0, 1:100, CH23009; Neuromics, Edina, MN, USA) or rabbit anti‐3‐nitrotyrosine (3‐NT, 1:200, AB5411; Millipore, Billerica, MA, USA) antibodies.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunolabeling