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 have previously reported that BMP-1 upregulates the expression of CCN2, which is well known as an osteogenesis/chondrogenesis marker, in 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: 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: The analysis of BMP-1-altered glycosylation profiles in insoluble fractions from hDPCs was performed by GlycoTechnica, Ltd. (Yokohama, Japan).

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: The analysis of BMP-1-altered glycosylation profiles in insoluble fractions from hDPCs was performed by GlycoTechnica, Ltd. (Yokohama, Japan).

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: The analysis of BMP-1-altered glycosylation profiles in insoluble fractions from hDPCs was performed by GlycoTechnica, Ltd. (Yokohama, Japan).

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

Journal: Scientific Reports

Article Title: Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

doi: 10.1038/s41598-023-30122-z

Figure Lengend Snippet: The CAMP promotes hDPC proliferation. ( A ) The cell viability of hDPCs against PAM. The hDPCs were grown for 15 min in the presence of PAM treated with CAMP at 30W (Low), 35W (Medium), and 40W (High) for 10, 20, and 40 s, respectively. The cells were replaced with fresh media and further grown for 18 h. MTT assay was performed as described in the method section. Ctrl represents non-treated hDPCs, whereas GAM indicates gas-activating media for 40 s. All data are shown as the mean ± SD of experiments conducted in triplicate. Statistical significance; ** P < 0.01. ( B ) Schematic of CAMP treatment in hDPCs and HaCaT cells.

Article Snippet: Human hair DPCs (hDPCs) were purchased from ScienCell (Cat. #2400, CA, USA).

Techniques: MTT Assay

Journal: Scientific Reports

Article Title: Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

doi: 10.1038/s41598-023-30122-z

Figure Lengend Snippet: PAM upregulates protein expression of the β-catenin signaling pathway and YAP/TAZ and enhances β-catenin translocation in hDPCs. ( A ) Representative immunoblots and ( B ) Relative fold-change expressions of β-catenin, cyclin D1, c-Myc, YAP, and TAZ in PAM-treated hDPCs, after 18 h of incubation. ( C ) Representative immunoblots and relative fold-change expression of p-β-catenin (S552)/β-catenin level in PAM-treated hDPCs, compared to gas control, at 15 min, 1 h, and 2 h of incubation times. ( D ) Representative images of β-catenin immunofluorescence staining and quantitative nuclear β-catenin fluorescence intensity of GAM and PAM against hDPCs for 15 min incubation. The cells were observed under a confocal microscope with 20 × magnification. The scale bar indicates a 50 µm distance. ( E ) The mRNA expressions of LEF1 genes in PAM-treated hDPCs, after 18 h of incubation. All data are shown as the mean ± SD of experiments conducted in triplicate. Statistical significance; ** P < 0.01; *** P < 0.001, and **** P < 0.0001. ns indicates not significant ( P > 0.05).

Article Snippet: Human hair DPCs (hDPCs) were purchased from ScienCell (Cat. #2400, CA, USA).

Techniques: Expressing, Translocation Assay, Western Blot, Incubation, Control, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

doi: 10.1038/s41598-023-30122-z

Figure Lengend Snippet: PAM impeded β-catenin degradation via USP47-regulated β-catenin ubiquitination in hDPCs. ( A ) The mRNA expressions of FER and USP47 genes in PAM-treated hDPCs, after 18 h of incubation. ( B ) Representative immunoblots and (C) Relative fold-change expressions of USP47 and p-β-catenin (Ser33/37/Thr41)/ β-catenin in PAM-treated hDPCs at 15 min, 1 h, and 2 h of incubation times. ( D ) The co-immunoprecipitation between p-β-catenin (Ser33/37/Thr41) and ubiquitin [Ub (HA)] in PAM-treated hDPCs at 15 min of incubation. ( E ) Representative images of USP47 and β-catenin immunofluorescence staining and quantitative analysis of USP47 and β-catenin co-localization in PAM-treated hDPCs compared to GAM control, after 15 min of incubation. The cells were observed under a confocal microscope with 20 × magnification. The scale bar indicates a 20 µm distance. All data are shown as the mean ± SD of experiments conducted in triplicate. Statistical significance; * P < 0.05; *** P < 0.001, and **** P < 0.0001. ns indicates not significant ( P > 0.05).

Article Snippet: Human hair DPCs (hDPCs) were purchased from ScienCell (Cat. #2400, CA, USA).

Techniques: Ubiquitin Proteomics, Incubation, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Control, Microscopy

Journal: Scientific Reports

Article Title: Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

doi: 10.1038/s41598-023-30122-z

Figure Lengend Snippet: PAM increases INPP5D mRNA expression and induces Akt and GSK-3β phosphorylation in hDPCs. (A) The heatmap results of human PI3K signaling TaqMan™ mRNA array of PAM-treated hDPCs. ( B ) The mRNA expression of INPP5D gene in PAM-treated hDPCs. ( C ) Representative immunoblots and relative fold-change expressions of p-Akt (Ser473)/ Akt in GAM and PAM- treated hDPCs. Cells were pre-treated with Akt1/2 kinase inhibitor (5 µM) for 1 h, and then treated with GAM or PAM for 15 min. The cell lysates were subjected to Western blot analysis. ( D ) Representative immunoblots and relative fold-change expressions of p-GSK-3α/β (Ser21/9) in GAM and PAM-treated hDPCs. All data are shown as the mean ± SD of experiments conducted in triplicate. Statistical significance; * P < 0.05; ** P < 0.01 and *** P < 0.001.

Article Snippet: Human hair DPCs (hDPCs) were purchased from ScienCell (Cat. #2400, CA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Scientific Reports

Article Title: Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

doi: 10.1038/s41598-023-30122-z

Figure Lengend Snippet: Conditioned media (CM) of hDPCs promotes protein expression of the β-catenin signaling pathway and YAP/TAZ in PAM-treated HaCaT cells. ( A ) The characteristics of hDPCs and HaCaT cells after 8 days of co-culturing and treating with PAM. The cell was visualized by Nikon Eclipse Ti-U inverted microscope with 4 × magnification. ( B ) The fluorescence images of GFP hDPCs and Hoechst 33,342 of the co-cultured PAM-treated hDPCs and HaCaT cells. The cells were observed under a confocal microscope with 10 × magnification. The scale bar indicates a 100 µm distance. ( C ) The mRNA expressions of versican genes in PAM-treated hDPCs, after 18 h of incubation. ( D ) Representative immunoblots and (E) Relative fold-change expressions of β-catenin, cyclin D1, c-Myc, YAP, and TAZ in HaCaT cells, after being treated with CM-derived hDPCs for 18 h. All data are shown as the mean ± SD of experiments conducted in triplicate. Statistical significance; * P < 0.05; ** P < 0.01, and *** P < 0.001.

Article Snippet: Human hair DPCs (hDPCs) were purchased from ScienCell (Cat. #2400, CA, USA).

Techniques: Expressing, Inverted Microscopy, Fluorescence, Cell Culture, Microscopy, Incubation, Western Blot, Derivative Assay

Journal: Scientific Reports

Article Title: Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

doi: 10.1038/s41598-023-30122-z

Figure Lengend Snippet: A schematic representation of the proposed molecular mechanisms of CAMP promoting hDPCs induction.

Article Snippet: Human hair DPCs (hDPCs) were purchased from ScienCell (Cat. #2400, CA, USA).

Techniques: