Journal: International journal of molecular sciences

Article Title: Fabrication of Hard Tissue Constructs from Induced Pluripotent Stem Cells for Exploring Mechanisms of Hereditary Tooth/Skeletal Dysplasia.

doi: 10.3390/ijms26020804

Figure Lengend Snippet: Figure 2. Effects of retinoic acid on expression of dentinogenesis- and osteogenesis-related genes of 3D hiPSC constructs during hard tissue induction. (a) Expression of phosphate metabolism- related genes such as dentin matrix acidic phosphoprotein 1 (DMP1) and phosphate regulating endopeptidase homolog X-linked (PHEX) was determined by quantitative real-time RT-PCR analysis (n = 3). (b) Expression of ECM-related genes such as dentin sialophosphoprotein (DSPP) and collagen type 1 alpha 1 (COL1A1) was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Gene expression of regulators of WNT signaling, such as WNT inhibitory factor 1 (WIF1) and sclerostin (SOST), was determined by quantitative real-time RT-PCR analysis (n = 3). (d) Gene expression of transcription factors such as msh homeobox 1 (MSX1), sp7 transcription factor (SP7), paired box gene 9 (PAX9), and LIM homeobox domain 6 (LHX6) was determined by quantitative real-time RT-PCR analysis (n = 3). Student’s t-test at the same time point. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Article Snippet: Next, the slides were incubated overnight at 4 ◦C with primary antibodies against DMP1 (Santa Cruz Biotechonology, Dallas, TX, USA), DSPP (Santa Cruz Biotechonology), WIF1 (Santa Cruz Biotechonology), and MSX1 (Santa Cruz Biotechonology), followed by incubation for 1 h with Pierce® Goat Anti-Rat IgG (H + L), Peroxidase Conjugated (Thermo Fisher Scientific) as the secondary antibody.

Techniques: Expressing, Construct, Quantitative RT-PCR, Gene Expression

Journal: International journal of molecular sciences

Article Title: Fabrication of Hard Tissue Constructs from Induced Pluripotent Stem Cells for Exploring Mechanisms of Hereditary Tooth/Skeletal Dysplasia.

doi: 10.3390/ijms26020804

Figure Lengend Snippet: Figure 3. Effects of retinoic acid on dentinogenesis- and osteogenesis-related protein expression of 3D hiPSC constructs after hard tissue induction. Three-dimensional hiPSC constructs were assessed on Day 20 of hard tissue induction with or without RA by immunohistochemical staining for DMP1, DSPP, WIF1, and MSX1 (scale bars: 100 µm).

Article Snippet: Next, the slides were incubated overnight at 4 ◦C with primary antibodies against DMP1 (Santa Cruz Biotechonology, Dallas, TX, USA), DSPP (Santa Cruz Biotechonology), WIF1 (Santa Cruz Biotechonology), and MSX1 (Santa Cruz Biotechonology), followed by incubation for 1 h with Pierce® Goat Anti-Rat IgG (H + L), Peroxidase Conjugated (Thermo Fisher Scientific) as the secondary antibody.

Techniques: Expressing, Construct, Immunohistochemical staining, Staining

Journal: International journal of molecular sciences

Article Title: Fabrication of Hard Tissue Constructs from Induced Pluripotent Stem Cells for Exploring Mechanisms of Hereditary Tooth/Skeletal Dysplasia.

doi: 10.3390/ijms26020804

Figure Lengend Snippet: Figure 5. Expression of dentinogenesis- and osteogenesis-related genes of 3D constructs fabricated from HPP patient-derived hiPSCs after hard tissue induction with RA. (a) Gene expression of DMP1 and PHEX was determined by quantitative real-time RT-PCR analysis (n = 3). (b) Gene expression of DSPP and COL1A1 was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Gene expression of WIF1 and SOST was determined by quantitative real-time RT-PCR analysis (n = 3). (d) Gene expression of MSX1, SP7, PAX9, and LHX6 was determined by quantitative real-time RT-PCR analysis (n = 3). Student’s t-test at the same time point. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Article Snippet: Next, the slides were incubated overnight at 4 ◦C with primary antibodies against DMP1 (Santa Cruz Biotechonology, Dallas, TX, USA), DSPP (Santa Cruz Biotechonology), WIF1 (Santa Cruz Biotechonology), and MSX1 (Santa Cruz Biotechonology), followed by incubation for 1 h with Pierce® Goat Anti-Rat IgG (H + L), Peroxidase Conjugated (Thermo Fisher Scientific) as the secondary antibody.

Techniques: Expressing, Construct, Derivative Assay, Gene Expression, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Fabrication of Hard Tissue Constructs from Induced Pluripotent Stem Cells for Exploring Mechanisms of Hereditary Tooth/Skeletal Dysplasia.

doi: 10.3390/ijms26020804

Figure Lengend Snippet: Figure 6. Expression of dentinogenesis- and osteogenesis-related proteins in 3D constructs fabricated from HPP-derived hiPSCs after hard tissue induction with RA. 3D hiPSC constructs were assessed on Day 20 of hard tissue induction with RA by immunohistochemical staining for DMP1, DSPP, WIF1, and MSX1 (scale bars: 100 µm).

Article Snippet: Next, the slides were incubated overnight at 4 ◦C with primary antibodies against DMP1 (Santa Cruz Biotechonology, Dallas, TX, USA), DSPP (Santa Cruz Biotechonology), WIF1 (Santa Cruz Biotechonology), and MSX1 (Santa Cruz Biotechonology), followed by incubation for 1 h with Pierce® Goat Anti-Rat IgG (H + L), Peroxidase Conjugated (Thermo Fisher Scientific) as the secondary antibody.

Techniques: Expressing, Construct, Derivative Assay, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: PRMT1-methylated MSX1 phase separates to control palate development

doi: 10.1038/s41467-025-56327-6

Figure Lengend Snippet: A Order/disorder region prediction of MSX1 by the protein sequence and disorder prediction (PONDR) algorithm. B , C Representative immunofluorescence images for endogenous MSX1 in HEK293T ( B ) and HEPM ( C ). Scale bars, 10 μm. D , E Representative confocal images of HEK293T ( D ) and HEPM ( E ) with ectopic overexpression of MSX1-mEGFP. Scale bars, 10 μm. F Representative fluorescence images of MSX1-mEGFP expressing HEK293T and HEPM treated with dimethyl sulfoxide (DMSO) or 5% 1,6-Hex (left), and quantification of liquid droplets formed in the indicated cells. Scale bars, 10 μm. HEK293T: n = 19. HEPM: −1,6-hex, n = 17; +1,6-hex, n = 13. G Representative graphs and quantitative analysis for the FRAP assay of MSX1-mEGFP condensates in HEK293T and HEPM. Scale bars, 10 μm. n = 3 biologically independent experiments. H Time-lapse micrographs of MSX1-mEGFP droplet fusion in HEPM. Scale bars, 10 μm. I Representative images of droplet formation of MSX1-FL-mEGFP and MSX1 IDR-mEGFP upon 3C cleavage. Scale bars, 10 μm. J Phase diagram of MSX1-mEGFP at different protein concentrations as a function of salt concentration, achieved by scoring for the existence of droplets or fibrillar hydrogels. Scale bars, 10 μm. K Representative images for the turbidity assay of MSX1-mEGFP protein solution with increasing protein concentration. L Representative images and quantification of MSX1-mEGFP and MSX1 protein solution treated with DMSO or 5% 1, 6-Hex. Scale bars, 10 μm. MSX1-mEGFP: n = 3, MSX1: n = 4 biologically independent experiments. M Representative droplet micrographs (top) and FRAP quantification (bottom) of MSX1-mEGFP protein in vitro. Scale bars, 10 μm. n = 4 biologically independent experiments. N Time-lapse micrographs of MSX1-mEGFP droplet fusion in vitro. Scale bars, 5 μm. O Schematic of MSX1 domains and truncated mutants. P Phase separation assay of truncated mutants of MSX1 in cells. Scale bars, 10 μm. Q Representative images of droplet formation for MSX1-FL-mEGFP or MSX1-ΔIDR-mEGFP at different protein concentrations in 25–200 mM NaCl. Scale bars, 10 μm. R Statistical analysis of the droplet formation of MSX1-FL-mEGFP or MSX1-ΔIDR-mEGFP. n = 3 biologically independent experiments. All data in this figure are represented as mean ± SD from at least three biologically independent experiments with similar results. Two-tailed Student’s t-test for ( F , L ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used for immunofluorescence and immunohistochemistry staining were specific for anti-SC-35 (Abcam, ab11826, 1: 50), anti-PML (Abcam, ab96051, 1:50), anti-Coilin (Abcam, ab11822, 1:50), anti-MSX1 (Bioss, bs-8512R, 1:50), anti-PRMT1 (Santa Cruz, sc-166963, 1:20), anti-GFP (Santa Cruz, sc-9996, 1:20), anti-PCNA (BOSTER, BM0104, 1:50), anti-PCNA (Proteintech, 60097-1-Ig, 1:50), anti-PHH3 (Santa Cruz, A2971, 1:20), anti-Vimentin (Cell signaling, 5741S, 1:50), anti-E-Cadherin (Cell signaling, 3195S, 1:1000), anti-Hexon (Bioss, bs-12354R, 1:50).

Techniques: Sequencing, Immunofluorescence, Over Expression, Fluorescence, Expressing, FRAP Assay, Concentration Assay, Protein Concentration, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: PRMT1-methylated MSX1 phase separates to control palate development

doi: 10.1038/s41467-025-56327-6

Figure Lengend Snippet: A Representative confocal images of MSX1-mEGFP-expressing HEK293T treated with DMSO or MS023 (left), and quantitative analysis of circularity (sphericity) of MSX1-mEGFP puncta per nucleus was shown (right). Scale bars, 10 μm. DMSO: n = 115, MS023: n = 216 condensates. B Representative confocal images (left) and circularity quantification (right) of MSX1-mEGFP-expressing HEK293T transfected with NC siRNA or PRMT1 siRNA. Scale bars, 10 μm. siNC: n = 105, siPRMT1: n = 310 condensates. C Representative images of FRAP of MSX1-mEGFP- expressing HEK293T treated with DMSO or MS023. Scale bars, 10 μm. D Representative graphs of FRAP of MSX1-mEGFP-expressing HEK293T transfected with NC or PRMT1 siRNA. Scale bars, 10 μm. E FRAP quantification of MSX1-mEGFP-expressing in HEK293T treated with DMSO or MS023. n = 3 biologically independent experiments. F Quantitative of FRAP assay in HEK293T transfected with NC or PRMT1 siRNA. n = 3 biologically independent experiments. G Representative immunofluorescence images for endogenous MSX1 and PRMT1 in HEK293T. Scale bars, 10 μm. H Representative western blot images of reciprocal Co-IP analysis confirming the association between MSX1 and PRMT1 in HEK293T ectopically expressing MSX1-mEGFP and PRMT1-FLAG. I Representative western blot images of pulldown assay using MSX1-mEGFP and PRMT1-mCherry purified proteins. J , K Confocal images ( J ) and colocalization analysis ( K ) of HEK293T with ectopic overexpression of MSX1-mEGFP and PRMT1-mCherry. Scale bars, 10 μm. The white solid line indicates the nucleus. L Representative DIC and fluorescence images of condensates formed by purified proteins MSX1-mEGFP and PRMT1-mCherry. Scale bars, 10 μm. M Representative western blot images of Co-IP assays assessing the aDMA and sDMA of MSX1. N Representative western blot images of Co-IP assays assessing the aDMA of MSX1 in treated with MS023 or PRMT1 siRNA. O Representative western blot images of in vitro methylation assay using MSX1 and PRMT1 purified proteins with/without S-adenosylmethionine (SAM) treatment. Coomassie blue-stained gel showed loading controls for MSX1 and PRMT1 purified proteins. P Droplet formation (left) and turbidity measurement (right) of unmethylated and methylated MSX1 purified proteins. Scale bars, 10 μm. n = 10 samples. All data in this figure are represented as mean ± SD from at least three biologically independent experiments with similar results. Two-tailed Student’s t-test for ( A , B , E , F , P ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used for immunofluorescence and immunohistochemistry staining were specific for anti-SC-35 (Abcam, ab11826, 1: 50), anti-PML (Abcam, ab96051, 1:50), anti-Coilin (Abcam, ab11822, 1:50), anti-MSX1 (Bioss, bs-8512R, 1:50), anti-PRMT1 (Santa Cruz, sc-166963, 1:20), anti-GFP (Santa Cruz, sc-9996, 1:20), anti-PCNA (BOSTER, BM0104, 1:50), anti-PCNA (Proteintech, 60097-1-Ig, 1:50), anti-PHH3 (Santa Cruz, A2971, 1:20), anti-Vimentin (Cell signaling, 5741S, 1:50), anti-E-Cadherin (Cell signaling, 3195S, 1:1000), anti-Hexon (Bioss, bs-12354R, 1:50).

Techniques: Expressing, Transfection, FRAP Assay, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Purification, Over Expression, Fluorescence, In Vitro, Methylation, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: PRMT1-methylated MSX1 phase separates to control palate development

doi: 10.1038/s41467-025-56327-6

Figure Lengend Snippet: A MSX1 PTM prediction. B Representative western blot images of aDMA-MSX1 levels in HEK293T transfected with indicated plasmids. C In vitro methylation assay of MSX1-FL/R150S/R157S-mEGFP purified proteins. D – G MSX1-FL-mEGFP, MSX1-R150S-mEGFP and MSX1-R157S-mEGFP phase separation in vivo. D Representative confocal images of condensates in HEK293T. Scale bars, 10 μm. E Circularity quantification of condensates in HEK293T. MSX1-FL: n = 83, MSX1-150S: n = 187, MSX1-157S: n = 128 condensates. F FRAP analysis of MSX1 condensates in HEK293T. Scale bars, 10 μm. G FRAP quantification of MSX1 condensates. n = 3 biologically independent experiments. H – K MSX1-FL-mEGFP, MSX1-R150S-mEGFP, and MSX1-R157S-mEGFP phase separation in vitro. H Representative confocal images of condensates formed by purified proteins. Scale bars, 10 μm. I Circularity quantification of condensates formed by purified proteins. FL: n = 873, R150S: n = 1602, R157S: n = 773 condensates. J Representative images of FRAP assays assessing time-dependent fluorescence recovery of condensates formed by purified proteins. Scale bars, 1 μm. K FRAP quantification of results condensates formed by purified proteins. n = 3 biologically independent experiments. L – O MSX1-FL-mEGFP, MSX1-R150S-mEGFP, MSX1-R157S-mEGFP, MSX1-R150F-mEGFP, MSX1-R157F-mEGFP, MSX1-R150K-mEGFP and MSX1-R157K-mEGFP phase separation in vivo and in vitro. L Representative confocal images of HEPM expressing MSX1-FL-mEGFP or its mutants. Scale bars, 10 μm. M Quantitative analysis of the area of MSX1-mEGFP puncta per nucleus. n = 5 fields. N Representative DIC images of condensates formed by purified proteins. Scale bars, 10 μm. O Quantification of condensate turbidity using purified proteins. n = 3 biologically independent experiments. P , Q MSX1-FL-mEGFP, MSX1-R150S-mEGFP, MSX1-R150S-mEGFP+PRMT1, MSX1-R157S-mEGFP and MSX1-R157S-mEGFP+PRMT1 phase separation in vivo. P Representative confocal images in HEK293T. Scale bars, 10 μm. Q Circularity quantification of condensates formed in HEK293T. FL: n = 44, R150S: n = 33, R150S + PRMT1: n = 43, R157S: n = 39, R157S + PRMT1: n = 36 condensates. R , S unmethylated or methylated MSX1-FL-mEGFP, MSX1-R150S-mEGFP, and MSX1-R157S-mEGFP phase separation in vitro. R Representative DIC and fluorescence images of condensates formed by purified proteins. Scale bars, 10 μm. S Quantification of condensate sphericity using purified proteins. unmeFL: n = 25, meFL: n = 25, unmeR150S: n = 27, meR150S: n = 23, unmeR157S: n = 20, meR157S: n = 20 condensates. All data in this figure are represented as mean ± SD from at least three biologically independent experiments with similar results. Two-tailed Student’s t-test for ( M , O ), One-way ANOVA with Dunnett’s multiple comparisons test for ( E , G , I , K ), One-way ANOVA with Turkey’s multiple comparisons test for ( Q , S ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used for immunofluorescence and immunohistochemistry staining were specific for anti-SC-35 (Abcam, ab11826, 1: 50), anti-PML (Abcam, ab96051, 1:50), anti-Coilin (Abcam, ab11822, 1:50), anti-MSX1 (Bioss, bs-8512R, 1:50), anti-PRMT1 (Santa Cruz, sc-166963, 1:20), anti-GFP (Santa Cruz, sc-9996, 1:20), anti-PCNA (BOSTER, BM0104, 1:50), anti-PCNA (Proteintech, 60097-1-Ig, 1:50), anti-PHH3 (Santa Cruz, A2971, 1:20), anti-Vimentin (Cell signaling, 5741S, 1:50), anti-E-Cadherin (Cell signaling, 3195S, 1:1000), anti-Hexon (Bioss, bs-12354R, 1:50).

Techniques: Western Blot, Transfection, In Vitro, Methylation, Purification, In Vivo, Fluorescence, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: PRMT1-methylated MSX1 phase separates to control palate development

doi: 10.1038/s41467-025-56327-6

Figure Lengend Snippet: A Sequence alignment of MSX1 orthologs in indicated vertebrates assessing the conservation of the PRMT1 methylation sites. B – G Control MO and prmt1 MO zebrafish embryos. B Representative images of phh3 staining in zebrafish embryos (left) and quantification of phh3 -positive cells in the ethmoid palate region of zebrafish embryos (right). Scale bars, 100 μm. n = 10 embryos. The white dashed line indicates the ethmoid palate. C Representative fluorescence ventral views of zebrafish embryos. The white solid line indicates the ethmoid palate. Scale bars, 500 μm. D Representative Alcian blue staining images of zebrafish embryos. The red dashed line indicates the ethmoid palate. Scale bars, 500 μm. E Quantification of the cleft palate rates in zebrafish embryos. n = 3 biologically independent experiments. F Representative images of zebrafish embryos expressing GFP-tagged MSX1 at 12 hpf in the ethmoid palate region. Scale bars, 20 μm. G Quantification of the circularity of MSX1 condensates in ethmoid palate region from zebrafish embryos. control : n = 152, prmt1 MO: n = 147 condensates. H – L Control MO, msx1 MO, and msx1 MO + PRMT1 mRNA zebrafish embryos. H Immunofluorescence staining images of phh3 in zebrafish embryos. The white dashed line indicates the ethmoid palate. Scale bars, 100 μm. I Quantitative analysis of phh3 -positive cells in the ethmoid palate region in zebrafish embryos. n = 27 embryos. J Fluorescence ventral views of zebrafish embryos. The white solid line indicates the ethmoid palate. Scale bars, 500 μm. K Alcian blue staining images of zebrafish embryos. The red dashed line indicates the ethmoid palate. Scale bars, 500 μm. L Quantification of the cleft palate rates in zebrafish embryos. n = 4 biologically independent experiments. M Representative western blot images of aDMA-MSX1 levels in zebrafish embryos microinjected with control and prmt1 MO. N Representative western blot images of aDMA-MSX1 levels in zebrafish embryos microinjected with MSX1-FL mRNA or its mutants, including R150S and R157S mRNA. O – S Control MO, msx1 MO, msx1 MO + FL mRNA, msx1 MO + R150S mRNA, and msx1 MO + R157S mRNA zebrafish embryos. O Representative immunofluorescence staining images of phh3 staining of zebrafish embryos. The white dashed line indicates the ethmoid palate. Scale bars, 100 μm. P Quantitative of phh3 -positive cells in the ethmoid palate region in zebrafish embryos. n = 21 embryos. Q Representative fluorescence ventral views of zebrafish embryos. The white solid line indicates the ethmoid palate. Scale bars, 500 μm. R Representative Alcian blue staining images of zebrafish embryos. The red dashed line represents the ethmoid palate. Scale bars, 500 μm. S Quantification of the cleft palate rates in zebrafish embryos. n = 3 biologically independent experiments. MO: morpholino, hpf: hours post-fertilization. All data in this figure are represented as mean ± SD from at least three biologically independent experiments with similar results. Two-tailed Student’s t-test for ( B , E , G , S ), One-way ANOVA with Dunnett’s multiple comparisons test for ( I , L ), and Turkey’s multiple comparisons test for ( P ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used for immunofluorescence and immunohistochemistry staining were specific for anti-SC-35 (Abcam, ab11826, 1: 50), anti-PML (Abcam, ab96051, 1:50), anti-Coilin (Abcam, ab11822, 1:50), anti-MSX1 (Bioss, bs-8512R, 1:50), anti-PRMT1 (Santa Cruz, sc-166963, 1:20), anti-GFP (Santa Cruz, sc-9996, 1:20), anti-PCNA (BOSTER, BM0104, 1:50), anti-PCNA (Proteintech, 60097-1-Ig, 1:50), anti-PHH3 (Santa Cruz, A2971, 1:20), anti-Vimentin (Cell signaling, 5741S, 1:50), anti-E-Cadherin (Cell signaling, 3195S, 1:1000), anti-Hexon (Bioss, bs-12354R, 1:50).

Techniques: Sequencing, Methylation, Control, Staining, Fluorescence, Expressing, Immunofluorescence, Western Blot, Two Tailed Test