Journal: International Journal of Molecular Sciences

Article Title: Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

doi: 10.3390/ijms20030551

Figure Lengend Snippet: Osteogenic differentiation of BMSCs and expression of m 6 A methyltransferase and demethylases. ( A , B ) The formation of mineralized nodules was analyzed in BMSCs undergoing osteogenic differentiation on Days 7, 14, and 21. The “white” scale bars represent 100 μm (original magnification ×100). ( C ) ALP activity assays. ALP activity was significantly higher in the cells cultured in osteogenic differentiation medium for seven days. ( D ) The expression of Runx2 , Alp and Ocn was assessed using qRT-PCR on Days 7 and 14 of culture in osteogenic induction medium. Gapdh was used as an internal control. ( E ) The expression of Runx2 and Osterix was examined using Western blotting. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( F , G ) RNA methylation modification-related enzymes were detected using qRT-PCR and Western blotting in cells cultured in osteogenic induction medium for 7 and 14 days. Gapdh was used as an internal control for qRT-PCR. Vinculin was used as an internal control in Western blotting. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The membrane was blocked in TBST containing 5% nonfat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: Mettl3 (1:1000; Proteintech, Chicago, IL, USA), Fto (1:1000; Abcam, Cambridge, UK), Alkbh5 (1:1000; Proteintech, Chicago, IL, USA), Runx2 (1:1000; Abcam, Cambridge, UK), Osterix (1:1000; Abcam, Cambridge, UK), Akt and P-Akt (1:1000; Cell Signaling Technology, Boston, MA, USA), Gapdh (1:1000; Abcam, Cambridge, UK), and Vinculin (1:1000; Cell Signaling Technology, Boston, MA, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Quantitative RT-PCR, Western Blot, Software, Methylation, Modification, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

doi: 10.3390/ijms20030551

Figure Lengend Snippet: Effect of Mettl3 knockdown on the osteogenic differentiation potential of BMSCs. ( A ) A green fluorescence protein marker was used to determine the transfer efficiency of Mettl3 knockdown in BMSCs. After transfection for 72 h, the cells were observed under a microscope (on the left). The right image is an immunofluorescence image taken at the same time. The “black” scale bars represent 100 μm (original magnification ×100). ( B ) The expression level of Mettl3 was determined using Western blotting in the Mettl3 -shRNA and Mettl3 -shCtrl groups. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( C ) The mRNA expression levels of Alp and Ocn in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using qRT-PCR after 7 and 14 days of osteogenic induction. Gapdh was used as an internal control. ( D ) The protein levels of Runx2 and Osterix in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using Western blotting after 7 and 14 days of osteogenic induction. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( E ) ALP activity was determined in Mettl3 -shRNA and Mettl3 -shCtrl cells cultured in osteogenic differentiation medium for seven days. ( F ) The formation of mineralized nodules was analyzed in the Mettl3 -shCtrl and Mettl3 -shRNA groups undergoing osteogenic induction on Days 7, 14, and 21. Mineralization was analyzed using Alizarin Red S staining. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The membrane was blocked in TBST containing 5% nonfat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: Mettl3 (1:1000; Proteintech, Chicago, IL, USA), Fto (1:1000; Abcam, Cambridge, UK), Alkbh5 (1:1000; Proteintech, Chicago, IL, USA), Runx2 (1:1000; Abcam, Cambridge, UK), Osterix (1:1000; Abcam, Cambridge, UK), Akt and P-Akt (1:1000; Cell Signaling Technology, Boston, MA, USA), Gapdh (1:1000; Abcam, Cambridge, UK), and Vinculin (1:1000; Cell Signaling Technology, Boston, MA, USA).

Techniques: Fluorescence, Marker, Transfection, Microscopy, Immunofluorescence, Expressing, Western Blot, shRNA, Software, Quantitative RT-PCR, Activity Assay, Cell Culture, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Gentamicin B1 is not a minor gentamicin component with major nonsense mutation suppression activity

doi: 10.1101/476564

Figure Lengend Snippet: Comparison of the PTC readthrough activity of B1-MCC and G418. DMS-114 cells were incubated with the indicated concentrations of B1-MCC sulfate and G418 sulfate for 72 h and the production of full-length p53 (FL-p53) and truncated p53 (TR-p53) was determined using automated capillary electrophoresis western analysis with vinculin as a protein loading control. Molecular weight markers are shown on the left in kDa.

Article Snippet: Capillary electrophoresis western analysis was carried out following the manufacturer’s instructions (ProteinSimple WES) using DO-1 p53 antibody from Santa Cruz Biotechnology and vinculin antibody (clone 728526) from R&D Systems.

Techniques: Comparison, Activity Assay, Incubation, Electrophoresis, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Gentamicin B1 is not a minor gentamicin component with major nonsense mutation suppression activity

doi: 10.1101/476564

Figure Lengend Snippet: Effect of gentamicin B1 on PTC readthrough in cells and in a cell-free assay. DMS-114 cells (A) and HDQ-P1 cells (B) were incubated with the indicated concentrations of G418 sulfate or gentamicin B1 acetate for 48 h (A) or 72 h (B) and production of full-length p53 (FL-p53) and truncated p53 (TR-p53) was determined using automated capillary electrophoresis western analysis. Vinculin was used as a protein loading control. (C) Synthetic R213X TP53 mRNA was subjected to in vitro translation in the presence of the indicated concentrations of G418 sulfate or gentamicin B1 acetate for 30 min and p53 was detected as in A and B. FL-p53 signal intensity relative to untreated control is shown below the lanes.

Article Snippet: Capillary electrophoresis western analysis was carried out following the manufacturer’s instructions (ProteinSimple WES) using DO-1 p53 antibody from Santa Cruz Biotechnology and vinculin antibody (clone 728526) from R&D Systems.

Techniques: Cell-Free Assay, Incubation, Electrophoresis, Western Blot, In Vitro

Journal: Scientific Reports

Article Title: Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration

doi: 10.1038/s41598-018-25668-2

Figure Lengend Snippet: Localization of RBM3 to SICs in different cell types and plating conditions. Images of RBM3 (green), F-actin (red, phalloidin), and DAPI-stained nuclei (blue) in B104 cells ( a–c ) and HeLa cells ( d–f ) 30 minutes after plating onto glass, collagen, and fibronectin. For each cell type and substrate in panels a through f, upper right sub-panels show close-ups of F-actin distribution in regions (hatched yellow rectangles) at cell margins that contain SICs; sub-panels in lower right show the overlay of RBM3 with F-actin. ( g–i ) Images of B104 cells grown on fibronectin that were labeled for RBM3 and other SIC components by immunofluorescence, and for tRNA by FISH: cells were double labeled for ( g ) RBM3 (green) and the SIC component vinculin (red); ( h ) FUS (green) and F-actin (red); and ( i ) RBM3 (green) and tRNA-glycine (red).

Article Snippet: Cells were incubated with a primary antibody dilution in 1% NGS 0.1% Triton X-100 for 1 hour at room temperature or 4 °C overnight (RBM3 1:1000, made in-house ; FUS, 1:200, Bethyl; vinculin 1:200, ProSci; myogenin 1:200, Santa Cruz; Histone 3p 1:200, EMD-Millipore; CRMP2 1:500, Abcam; FLAG 1:1000, Sigma; Vimentin 1:500, Sigma) and rinsed 3 times with PBS 0.1%- Triton X-100.

Techniques: Staining, Labeling, Immunofluorescence

Journal: Scientific Reports

Article Title: Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration

doi: 10.1038/s41598-018-25668-2

Figure Lengend Snippet: RBM3-regulated changes in cell morphology involve RhoA-ROCK signaling. ( a ) Western blots showing expression levels of the indicated proteins at 0, 60, and 120 minutes after replating of B104 cells maintained in control (con), RBM3 knockdown (si), and RBM3 overexpression (o/x) conditions. A large increase in RhoA is observed at all timepoints in RBM3 knockdown cells, along with tubulin and a modest increase in vinculin, an SIC component. CRMP2, rhotekin, and CDC42 were downregulated in the knockdown condition. In cells overexpressing RBM3, RhoA was initially downregulated. ( b ) Bar graph of changes in RhoA, CRMP2 and vinculin at 2 hrs in RBM3 knockdown vs control cells (n = 4 experiments; *p < 0.05, 1-sample t-test), normalized to eIF6 (present blots) or β-actin (additional experiments, Supplementary Fig. ). ( c–f ) Images of RBM3 (green), F-actin (red, phalloidin), and nuclei (blue) in B104 cells under control (con) and RBM3 knockdown conditions (si), with and without the ROCK inhibitor Y27632 (100 μM). Inhibition of ROCK rescued cell polarity in RBM3 knockdown B104 cells (scale bar = 10 μm). ( g , h ) Graphs summarizing cell body areas ( g ) and major process lengths ( h ) at 2 and 24 hrs post replating in the following treatment conditions: control cells (con: n = 17 at 2 hrs; n = 23 at 24 hrs), RBM3 knockdown cells (si: n = 22 at 2 hrs; n = 20 at 24 hrs), control cells treated with Y27632 (con + Y27632: n = 15 at 2 hrs; n = 25 at 24 hrs), RBM3 knockdown cells treated with Y27632 (si + Y27632: n = 12 at 2 hrs; n = 15 at 24 hrs; *p < 0.05, **p < 0.005, ***p < 0.0005; t-tailed t-test). Cells in which RBM3 was knocked down, but not treated with Y27632, did not have processes to measure.

Article Snippet: Cells were incubated with a primary antibody dilution in 1% NGS 0.1% Triton X-100 for 1 hour at room temperature or 4 °C overnight (RBM3 1:1000, made in-house ; FUS, 1:200, Bethyl; vinculin 1:200, ProSci; myogenin 1:200, Santa Cruz; Histone 3p 1:200, EMD-Millipore; CRMP2 1:500, Abcam; FLAG 1:1000, Sigma; Vimentin 1:500, Sigma) and rinsed 3 times with PBS 0.1%- Triton X-100.

Techniques: Western Blot, Expressing, Control, Knockdown, Over Expression, Inhibition

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Therapeutic Angiogenesis by Implantation of a Capillary Structure Constituted of Human Adipose Tissue Microvascular Endothelial Cells

doi: 10.1161/atvbaha.109.198994

Figure Lengend Snippet: Figure 5. Effect of the ROCK inhibitor Y-27632 on S1P-treated HOMECs. A, Transwell migration assay of HOMECs (n15 HPFs). Bars indicate SDs. B through D, Immunocytochemistry of HOMECs stained for vinculin (green), F-actin (red), and nuclei (blue). Arrowheads, focal adhesion plaque; arrows, cortical actin ring; asterisks, lamellipodia. E, Cortical actin ring formation in HOMECs treated with vehi- cle, S1P, or S1PY-27632 (n10, 18, and 13 HPFs, respectively). Bars indicate SDs.

Article Snippet: Hanks’ balanced salt solution (HBSS) and bovine serum albumin (BSA) were purchased from Wako (Osaka, Japan); M199, MCDB131, and OptiMEM were from Gibco (Grand Island, NY); fetal bovine serum (FBS) was obtained from Bioserum (Canterbury, Australia); anti-human vinculin mouse moAb (clone V284) was from Acris GmbH (Hiddenhausen, Germany); anti-human CD31 mouse monoclonal antibody was from DAKO (Glostrup, Denmark); anti-human eNOS and VE-cadherin mouse monoclonal antibodies were from BD Pharmingen (San Diego, CA); control mouse IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Alexa-488 conjugated goat anti-mouse IgG, TO-PRO3, Hoechst 33342, and Lipofectamine RNAiMAX reagent were from Invitrogen Life Technologies (Carlsbad, CA); anti-human S1P1, S1P2, or S1P3 rabbit poAbs were from MBL (Woburn, MA); PKH26GL, radio immunoprecipitation assay (RIPA) buffer, aprotinin, leupeptin, pepstatin A, anti-β-actin moAb, and TRITC-phalloidin were from Sigma (St. Louis, MO); Bio-Rad Dc Protein Assay Kit and horseradish peroxidase (HRP)-conjugated protein G antibody were from Bio-Rad (Hercules, CA); PVDF membrane and ECL plus Western blotting detection reagents were from GE Healthcare (Buckinghamshire, UK); First-strand cDNA synthesis kit for RT-PCR and Light Cycler System were from Roche (Mannheim, Germany); SYBR Green I Premix Ex Taq was from TAKARA (Shiga, Japan); Sphingosine-1-phosphate (S1P) and Y-27632, an inhibitor of Rho-associated kinase (ROCK) were purchased from Biomol (Plymouth Meeting, PA).

Techniques: Transwell Migration Assay, Immunocytochemistry, Staining