human dental pulp cells (hdpcs)  (Lonza)

Journal: Journal of Cell Communication and Signaling

Article Title: BMP-1-induced GBA1 nuclear accumulation provokes CCN2 mRNA expression via importin-β-mediated nucleocytoplasmic pathway

doi: 10.1007/s12079-023-00740-3

Figure Lengend Snippet: A comprehensive analysis of BMP-1-altered glycosylation profiles in hDPCs. A The volcano plot represents fold change (x-axis) and p-values (y-axis) of signal intensities of each lectin between the absence or presence of rhBMP-1 (500 ng/mL) for 1 h. Sambucus nigra agglutinin (SNA), Sambucus sieboldiana agglutinin (SSA), and Trichosanthes japonica agglutinin-I (TJA-I), which commonly recognize α2,6-sia, showed significant reductions in signals. Results are from three different donors. Significant differences in lectin intensity were calculated by F-test. B A lectin-probed blotting analysis of the insoluble fraction (10 µg protein) from hDPCs. Nitrocellulose membranes were incubated with HRP-conjugated SNA followed by visualization. As expected, the signal of α2,6-sia was attenuated by BMP-1 compared to controls. C The enrichment of α2,6-sialylated glycoprotein from the insoluble fraction of hDPCs. After cell harvesting, the isolated insoluble fraction samples were suspended in adsorption buffer and applied to an SNA lectin column. After washing two times with adsorption buffer, SNA binding glycoproteins were eluted two times from the affinity column with elution buffer (sialyl-lactose). After elution, the column was washed again two times with adsorption buffer, and the purity of serial wash fractions and the eluate was assessed by SDS-PAGE with probing using HRP-conjugated SNA. D The eluate was desalted and concentrated by diafiltration. The concentrate (Conc.) and the filtrate (Filt.) were separated by SDS-PAGE and stained with CBB. Std. denotes protein molecular weight standards

Article Snippet: We previously showed that the expression of the osteogenesis marker cellular communication network factor (CCN) 2 was increased by the dynamin-dependent endocytosis of BMP-1 in human dental pulp cells (hDPCs) (Muromachi et al. 2015 ).

Techniques: Glycoproteomics, Incubation, Cell Harvesting, Isolation, Adsorption, Binding Assay, Affinity Column, SDS Page, Diafiltration Assay, Staining, Molecular Weight

Journal: Journal of Cell Communication and Signaling

Article Title: BMP-1-induced GBA1 nuclear accumulation provokes CCN2 mRNA expression via importin-β-mediated nucleocytoplasmic pathway

doi: 10.1007/s12079-023-00740-3

Figure Lengend Snippet: Inhibition of BMP-1-induced CCN2 expression in hDPCs transfected with GBA1 siRNAs. A Protein levels of GBA1 and β-actin were detected by Western blotting in the cells transfected with GBA1 or scramble siRNAs. Cell lysates (5 µg protein) were used for immunoblotting. B Densitometric quantification of immunoreactive bands was calculated as the ratio of GBA1 to β-actin and is normalized against control. C BMP-1-induced CCN2 mRNA expression was attenuated in the cells transfected with siRNAs for GBA1 as compared to in scramble siRNA-transfected cells. Results are presented as the means ± SE. Statistical analysis was performed by Tukey’s test. *P < 0.05

Article Snippet: We previously showed that the expression of the osteogenesis marker cellular communication network factor (CCN) 2 was increased by the dynamin-dependent endocytosis of BMP-1 in human dental pulp cells (hDPCs) (Muromachi et al. 2015 ).

Techniques: Inhibition, Expressing, Transfection, Western Blot, Control

Journal: Journal of Cell Communication and Signaling

Article Title: BMP-1-induced GBA1 nuclear accumulation provokes CCN2 mRNA expression via importin-β-mediated nucleocytoplasmic pathway

doi: 10.1007/s12079-023-00740-3

Figure Lengend Snippet: BMP-1 regulates the nuclear accumulation of GBA1 in hDPCs. A To confirm the results of MS analysis, the insoluble fractions from hDPCs were analyzed by Western blotting with rabbit anti-GBA1 antibody. B BMP-1 facilitates the nuclear accumulation of GBA1. Cells were incubated with rhBMP-1 (500 ng/mL) for 5, 15, 30, 60, and 180 min. After fixation by paraformaldehyde, the signals of GBA1 were observed using a confocal laser scanning microscope. Pictures indicate GBA1 (Alexa fluor 594, red), filamentous actin (Alexa Fluor 488 phalloidin, green), and nuclei (DAPI, blue). Scale bars: 20 μm. Negative controls were treated without the primary antibody. C GBA1 signals were observed in three randomly chosen sites per three different experiments (n = 3). GBA1 signal intensity in the nuclei was quantified by measuring all of the nuclei in the pictures. Results are presented as the means ± SE. Statistical analysis was performed by Tukey’s test. **P < 0.01 versus control. D Orthogonal view from confocal z-stack images of hDPCs. hDPCs were incubated in the presence of rhBMP-1 (500 ng/mL) for 1 h. Pictures indicate GBA1 (Alexa fluor 594, red) and nuclei (DAPI, blue). Blue lines indicate the X/Y plane, the green line indicates the X/Z plane, and the red line indicates the Y/Z plane. E A typical image of GBA1 and nuclei from (D). GBA1 signals were observed in the low-density region of DAPI-staining (white arrowheads). (F) Cytoplasmic GBA1 is localized in lysosomes, and it also accumulates in the nucleus in the presence of BMP-1. hDPCs were incubated in the absence or presence of rhBMP-1 (500 ng/mL) for 1 h. After fixation by ice-cold methanol, the localization of GBA1 was observed using a confocal laser scanning microscope. Pictures indicate GBA1 (Alexa fluor 594, red), LAMP1 (Alexa Fluor 488, green), nuclei (DAPI, blue), and differential interference contrast (DIC) images. Scale bars: 20 μm

Article Snippet: We previously showed that the expression of the osteogenesis marker cellular communication network factor (CCN) 2 was increased by the dynamin-dependent endocytosis of BMP-1 in human dental pulp cells (hDPCs) (Muromachi et al. 2015 ).

Techniques: Western Blot, Incubation, Laser-Scanning Microscopy, Control, Staining