Journal: Cell death and differentiation

Article Title: Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis.

doi: 10.1038/s41418-022-00956-7

Figure Lengend Snippet: Fig. 2 USP49 upregulates endogenous PAX9 and MSX1 proteins. A Validation of the efficiency of sgRNAs were carried out in hDPSCs by transiently transfecting sgRNA1 to sgRNA4 targeting USP49, and immunoblotting with the USP49 antibody. The presented immunoblots are representative of three independent experiments (n = 3). B Validation of the efficiency of shRNAs were carried out in hDPSCs by transiently transducing shRNA1 and shRNA2 targeting USP49, and immunoblotting with the USP49 antibody. The presented immunoblots are representative of three independent experiments (n = 3). C hDPSCs were transfected with two best sgRNAs (sgRNA1 and sgRNA3) and one shRNA (shRNA1) targeting USP49 to check the endogenous protein levels of USP49, PAX9, and MSX1 using their respective endogenous antibodies. The presented immunoblots are representative of three independent experiments (n = 3). D hDPSCs were transfected with increasing concentrations of Flag-USP49 to check the endogenous USP49, PAX9, and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). E hDPSCs were transfected with increasing concentrations of Flag-USP49CA to check the endogenous USP49, PAX9, and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). F Reconstitution effect of USP49 on endogenous PAX9 or MSX1 in USP49-depleted cells. Protein expression was analyzed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. The presented immunoblots are representative of three independent experiments (n = 3).

Article Snippet: MATERIALS AND METHODS Plasmids, sgRNAs, and shRNAs A mammalian expression vector encoding Myc-Flag-tagged PAX9 was purchased from OriGene (Cat. no. RC200380, Rockville, USA).

Techniques: Biomarker Discovery, Western Blot, Transfection, shRNA, Expressing, Control

Journal: Journal of dental research

Article Title: Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

doi: 10.1177/0022034517729322

Figure Lengend Snippet: Figure 1. Clinical characteristics of probands with a PAX9 mutation. (A) Intraoral photos and panoramic radiographs of probands with distinct PAX9 mutations. Asterisks represent the position of missing teeth. (B) The prevalence of missing teeth in all PAX9 mutation patients (n = 16). L, lower; U, upper. (C) The prevalence of microdontia in all patients with a PAX9 mutation.

Article Snippet: Construction of PAX9 Expression Vectors and Site-Directed Mutagenesis The human PAX9 complementary DNA (cDNA) (accession number: NM_006194; Origene) was subcloned into the pCMVC-Myc expression vector (Beyotime) between 5′-BamHI and 3′-EcoRI sites to generate the pCMV-PAX9-Myc wild-type plasmid.

Techniques: Mutagenesis

Journal: Journal of dental research

Article Title: Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

doi: 10.1177/0022034517729322

Figure Lengend Snippet: Figure 2. Location and conservation analysis of tooth agenesis associated PAX9 mutations. (A) Distribution of mutations identified in patients with tooth agenesis in the PAX9 protein. Reported mutations are labeled in blue. Black indicates novel PAX9 mutations. (B) Schematic diagram of the PAX9 gene. (C) Conservation analysis of affected amino acids in the PAX9 protein among 9 different vertebrate species.

Article Snippet: Construction of PAX9 Expression Vectors and Site-Directed Mutagenesis The human PAX9 complementary DNA (cDNA) (accession number: NM_006194; Origene) was subcloned into the pCMVC-Myc expression vector (Beyotime) between 5′-BamHI and 3′-EcoRI sites to generate the pCMV-PAX9-Myc wild-type plasmid.

Techniques: Labeling

Journal: Journal of dental research

Article Title: Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

doi: 10.1177/0022034517729322

Figure Lengend Snippet: Figure 3. Functional studies of mutant PAX9 proteins. (A) Expression of wild-type and mutant PAX9 proteins detected by Western blot. (B) Relative messenger RNA (mRNA) expression levels of mutant PAX9 at 12 h posttransfection. (C) After treatment with actinomycin D, PAX9 mRNAs underwent decay. The mRNA stability is presented by the percentage of remaining mRNAs at 4 and 8 h posttreatment. *P < 0.05. (D) Subcellular localization of wild-type and mutant PAX9 proteins assessed by immunofluorescence. (E) The transcriptional activation abilities of PAX9 mutants on the BMP4 promoter assessed by luciferase reporter assay. **P < 0.01. (F) Binding of PAX9 mutants to the paired domain consensus sequence CD19-2 (A-ins) assessed by an electrophoretic mobility shift assay.

Article Snippet: Construction of PAX9 Expression Vectors and Site-Directed Mutagenesis The human PAX9 complementary DNA (cDNA) (accession number: NM_006194; Origene) was subcloned into the pCMVC-Myc expression vector (Beyotime) between 5′-BamHI and 3′-EcoRI sites to generate the pCMV-PAX9-Myc wild-type plasmid.

Techniques: Functional Assay, Mutagenesis, Expressing, Western Blot, Immunofluorescence, Activation Assay, Luciferase, Reporter Assay, Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay

Journal: Medicina

Article Title: PAX7 , PAX9 and RYK Expression in Cleft Affected Tissue

doi: 10.3390/medicina57101075

Figure Lengend Snippet: Immunoreactive structures for paired box 9 ( PAX9 ) in cleft affected tissue. ( A ) Control group tissue with a moderate number of PAX9 -containing epitheliocytes in the surface epithelium and barely detectable PAX9 -positive cells in the connective tissue, PAX9 IMH, 200×. ( B ) Unilateral cleft lip affected tissue with moderate number of PAX9 epitheliocytes in the patient epithelium and a few PAX9 -containing structures in the connective tissue, PAX9 IMH, 200×. ( C ) Bilateral cleft lip affected tissue with a few PAX9 -positive structures in the surface epithelium and a few PAX9 -containing cells in the connective tissue, PAX9 IMH, 200×. ( D ) Isolated cleft palate affected tissue with a moderate number of PAX9 -positive epitheliocytes in the surface epithelium and a few PAX9 -positive structures in the connective tissue, PAX9 IMH, 200×.

Article Snippet: Later, deparaffinization was carried out and further slide staining was performed with the biotin-streptavidin immunohistochemical method for detection of the presence of specific proteins within the tissue with antibodies for PAX7 (ab55494, 1:100, Abcam, Cambridge, UK), PAX9 (orb11242, 1:100, Biorbyt Ltd., Cambridge, UK), and RYK (orb38371, 1:100, Biorbyt Ltd., Cambridge, UK).

Techniques: Control, Isolation

Journal: Medicina

Article Title: PAX7 , PAX9 and RYK Expression in Cleft Affected Tissue

doi: 10.3390/medicina57101075

Figure Lengend Snippet: Summary of semiquantitative evaluation showing the median values of PAX7 , PAX9 , and RYK immunoreactivity in the control group, unilateral cleft lip patient group, bilateral cleft lip patient group, and isolated cleft palate patient group.

Article Snippet: Later, deparaffinization was carried out and further slide staining was performed with the biotin-streptavidin immunohistochemical method for detection of the presence of specific proteins within the tissue with antibodies for PAX7 (ab55494, 1:100, Abcam, Cambridge, UK), PAX9 (orb11242, 1:100, Biorbyt Ltd., Cambridge, UK), and RYK (orb38371, 1:100, Biorbyt Ltd., Cambridge, UK).

Techniques: Control, Isolation

Journal: Medicina

Article Title: PAX7 , PAX9 and RYK Expression in Cleft Affected Tissue

doi: 10.3390/medicina57101075

Figure Lengend Snippet: Correlations between paired box 7 ( PAX7 ), paired box 9 ( PAX9 ), and receptor-like tyrosine kinase ( RYK ) immunopositive structures in unilateral cleft lip affected tissue based on Spearman’s rank correlation coefficient calculation (r s —Spearman’s rho value).

Article Snippet: Later, deparaffinization was carried out and further slide staining was performed with the biotin-streptavidin immunohistochemical method for detection of the presence of specific proteins within the tissue with antibodies for PAX7 (ab55494, 1:100, Abcam, Cambridge, UK), PAX9 (orb11242, 1:100, Biorbyt Ltd., Cambridge, UK), and RYK (orb38371, 1:100, Biorbyt Ltd., Cambridge, UK).

Techniques:

Journal: Medicina

Article Title: PAX7 , PAX9 and RYK Expression in Cleft Affected Tissue

doi: 10.3390/medicina57101075

Figure Lengend Snippet: Correlations between paired box 7 ( PAX7 ), paired box 9 ( PAX9 ), and receptor-like tyrosine kinase ( RYK ) immunopositive structures in bilateral cleft lip affected tissue based on Spearman’s rank correlation coefficient calculation (r s —Spearman’s rho value).

Article Snippet: Later, deparaffinization was carried out and further slide staining was performed with the biotin-streptavidin immunohistochemical method for detection of the presence of specific proteins within the tissue with antibodies for PAX7 (ab55494, 1:100, Abcam, Cambridge, UK), PAX9 (orb11242, 1:100, Biorbyt Ltd., Cambridge, UK), and RYK (orb38371, 1:100, Biorbyt Ltd., Cambridge, UK).

Techniques:

Journal: Medicina

Article Title: PAX7 , PAX9 and RYK Expression in Cleft Affected Tissue

doi: 10.3390/medicina57101075

Figure Lengend Snippet: Correlations between paired box 7 ( PAX7 ), paired box 9 ( PAX9 ), and receptor-like tyrosine kinase ( RYK ) immunopositive structures in isolated cleft palate affected tissue based on Spearman’s rank correlation coefficient calculation (r s —Spearman’s rho value).

Article Snippet: Later, deparaffinization was carried out and further slide staining was performed with the biotin-streptavidin immunohistochemical method for detection of the presence of specific proteins within the tissue with antibodies for PAX7 (ab55494, 1:100, Abcam, Cambridge, UK), PAX9 (orb11242, 1:100, Biorbyt Ltd., Cambridge, UK), and RYK (orb38371, 1:100, Biorbyt Ltd., Cambridge, UK).

Techniques: Isolation

Journal: Clinical Epigenetics

Article Title: Transcriptional epigenetic regulation of Fkbp1 / Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

doi: 10.1186/s13148-021-01149-8

Figure Lengend Snippet: Bisulfite sequencing of PAX9 and FKBP1B regulatory CGIs. Representation of ​​bisulfite-modified DNA fragment of both candidate genes from the sensitive and resistant tumor lines A2780 and OVCAR3, as well as DNA obtained from healthy ovarian tissue (OC) from patients undergoing sex change, DNA from human peripheral blood mononuclear cells (PBMCs) and from the tumor lines HeLa, BT747, LoVo and PC3. Samples were sequenced with sense primer for PAX9 and antisense for FKBP1B except in OVCAR3-S. Asterisks indicate methylated positions

Article Snippet: Probes for gene expression (Applied Biosystems, USA) were as follows: PAX9: Hs00196354_m1; FKBP1B : Hs00997682_m1; and GAPDH: Hs03929097_g1.

Techniques: Methylation Sequencing, Modification, Methylation

Journal: Clinical Epigenetics

Article Title: Transcriptional epigenetic regulation of Fkbp1 / Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

doi: 10.1186/s13148-021-01149-8

Figure Lengend Snippet: Absolute methylation and relative expression levels for FKBP1B and PAX9 and the effect of overexpression of candidate genes on cell sensitivity to CDDP in the OVCAR3 cell line. A , B Representation of methylated and unmethylated molecules assessed through qMSP (quantitative methylation-specific PCR). The methylation rate obtained from three replicates for each type of evaluated sample is shown. Assays were performed in all experimental conditions: S, R and RT. S, sensitive; R, resistant; RT, resistant treated with epigenetic reactivation drugs (5-Aza and TSA). Each trial was repeated twice, and a non-neoplastic ovary sample was included as the control. C , D The expression levels of each candidate gene assessed by qRT-PCR were normalized using GAPDH as the endogenous control. Assays were performed in all experimental conditions of the OVCAR3 cell line. Data are represented in the log10 scale using the sensitive experimental group as a calibrator. Bars represent the mean ± SE of two independent experiments performed in duplicate. ** p ≤ 0.01 *** p ≤ 0.001. E Viability assays of the OVCAR3 cell lines transfected with pCMV6 (S-MOCK and R-MOCK) and with the overexpression vectors (R-PAX9 and R-FKBP1B). Each experimental group was exposed for 48 h to six different CDDP concentrations, and the data were normalized to each untreated control (set to 100%). The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each analyzed cell line. P-values < 0.01 indicated a significant change in drug sensitivity (Student’s t-test)

Article Snippet: Probes for gene expression (Applied Biosystems, USA) were as follows: PAX9: Hs00196354_m1; FKBP1B : Hs00997682_m1; and GAPDH: Hs03929097_g1.

Techniques: Methylation, Expressing, Over Expression, Control, Quantitative RT-PCR, Transfection, Concentration Assay

Journal: Clinical Epigenetics

Article Title: Transcriptional epigenetic regulation of Fkbp1 / Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

doi: 10.1186/s13148-021-01149-8

Figure Lengend Snippet: FKBP1B and PAX9 methylation analysis in primary tumors and survival analysis. A The methylation rates were evaluated in clinical samples from different cohorts through MSP (methylation-specific PCR). For each sample, the presence of a product in lane M was considered as methylated DNA, while the product amplification in lane U was considered as non-methylated. Negative and positive controls for each reaction corresponded to DNA from PBMCs and in vitro methylated DNA, respectively. Representative MSPs of candidate genes are shown. B Samples 78, 79, 80, 82 and 140 showed methylation for FKBP1B, C and samples 152, 153 and 155 showed methylation for PAX9 . Kaplan–Meier comparison in terms of overall survival (OS) between cisplatin response and PAX9 methylation in 21 patients with platinum-resistant ovarian cancer treated. Qualitative and quantitative methylation comparisons correspond to D and E , respectively. Log-rank tests were used for comparisons, and p values < 0.05 indicated a significant change in OS. We employed a Kaplan–Meier plotter tool to compare PAX9 expression versus OS and progression-free survival (PFS) ( F , G ) in 505 and 475 selected TCGA patients, respectively. A log-rank test was applied for comparisons, and p values < 0.05 indicated a significant change in OS

Article Snippet: Probes for gene expression (Applied Biosystems, USA) were as follows: PAX9: Hs00196354_m1; FKBP1B : Hs00997682_m1; and GAPDH: Hs03929097_g1.

Techniques: Methylation, Amplification, In Vitro, Comparison, Expressing

Journal: Stem cells international

Article Title: Efficient Differentiation of Mouse Induced Pluripotent Stem Cells into Alveolar Epithelium Type II with a BRD4 Inhibitor.

doi: 10.1155/2019/1271682

Figure Lengend Snippet: Figure 1: (a) Differentiation protocols for the derivation of ATII using an embryoid body seeding method and a stepwise differentiation method. (b) Quantitative polymerase chain reaction analysis of DE (day 7) (n = 5). Expression of Cxcr4, Foxa2, and Sox17 mRNA. Expression ratios were normalized to the level of Gapdh expression. Expression levels were compared with those of undifferentiated iPSCs (day 0). ∗P < 0:05. (c) Phase-contrast image and immunofluorescence images of FOXA2 in DE (day 7). Scale bars: 100 μm. (d) Quantitative polymerase chain reaction analysis of AFE (day 9) (n = 4). Expression of Pax9, Tbx1, and Sox2 mRNA. Expression ratios were normalized to the level of Gapdh expression. Expression levels were compared with those of undifferentiated iPSCs (day 0). ∗P < 0:05 . (e) Phase-contrast image and immunofluorescence images of PAX9 and SOX2 in AFE (day 9). Scale bars: 100 μm.

Article Snippet: The TaqMan Gene Expression Assay identifiers of detected genes were Mm01996749_s1 (Cxcr4), Mm01976556_s1 (Foxa2), Mm00488363_m1 (Sox17), Mm03053810_s1 (Sox2), Mm00440629_m1 (Pax9), Mm00448949_m1 (Tbx1), Mm00447558_m1 (Nkx2-1), Mm00485928_m1 (Atxn1), Mm00488144_m1 (Sftpc), Mm00455678_m1 (Sftpb), Mm03053917_m1 (Klf4), Mm00487804_m1 (Myc), Mm03053917_g1 (Pou5f1), and Mm99999915_g1 (Gapdh).

Techniques: Real-time Polymerase Chain Reaction, Expressing