Journal: Nature Communications

Article Title: Autocrine ECM molecules establish MSC quiescence during incisor development by disrupting WNT ligand trafficking process

doi: 10.1038/s41467-025-65705-z

Figure Lengend Snippet: a Expression of active β-Catenin and its downstream targets c-Myc, Cyclin D1, and LEF1 in DPCs after Smoc1 or Smoc2 overexpression, as assessed by western blot. b Expression level of active β-Catenin and its downstream targets c-Myc, and Cyclin D1 in DPCs after Smoc1/2 overexpression alone or simultaneously. c Immunostaining of β-Catenin in control, Wnt agonist WAY and WAY with Smoc1/2 treated groups. Scale bars: 10 μm. Representative images from three independent experiments with similar results. SMOC1/2 interfere with Wnt10a transportation. d The schematic drawing illustrates the experimental scheme of DPCs supplied with beads. Created with MedPeer (medpeer.cn). e Immunostaining of Wnt10a in cultured DPCs supplied with beads. White arrows indicate WNT Dynabeads. Scale bars: 10 μm. Representative images from three independent experiments with similar results. f Expression of active β-Catenin and its downstream targets c-Myc, Cyclin D1, and LEF1 in DPCs after Gpc6 silencing, as assessed by western blot. g Immunostaining of β-Catenin in siRNA negative control (si NC ), WAY with si NC and WAY with si Gpc6 treated groups. Scale bars: 10 μm. Representative images from three independent experiments with similar results. h Co-IP of GPC6 with SMOC1 or SMOC2 in DPCs. i Co-localization analysis of SMOC1/2 with GPC6, as assessed by immunostaining. White arrows indicate co-localization signals. Scale bars: 10 μm. Representative images from three independent experiments with similar results. j Co-IP of GPC6 with WNT10a in DPCs in control , Smoc2 or Smoc1 overexpression groups. Source data are provided as a Source Data file. k Mechanism diagram. SMOC1/2 inhibit WNT trafficking by competitively binding to GPC6, thereby disrupting WNT ligand distribution within the niche. Created with MedPeer (medpeer.cn).

Article Snippet: The anti-Smoc1 Mouse mAb (Santa Cruz Biotechnology, sc-390448), anti-Smoc2 Mouse mAb (Santa Cruz Biotechnology, sc376104), and anti-Gpc6 Rabbit mAb (OriGene, TA351222S) were used for immunoprecipitation of Smoc1, Smoc2, and Gpc6 proteins, respectively.

Techniques: Expressing, Over Expression, Western Blot, Immunostaining, Control, Cell Culture, Negative Control, Co-Immunoprecipitation Assay, Binding Assay

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: High glucose stress activates the mitochondrial repressor MTERF3. A) Quantification of the MTERF3 (Mitochondrial Transcription Termination Factor 3) RNA transcript by taqman quantitative PCR in HDF grown in 5.55 mM or 12 mM glucose. Normalization of the data was performed using GusB (β-glucuronidase) (N = 3). B) Determination by Simple WES of MTERF3 protein expression in HDF cultivated 48H in DMEM with 5.55 mM or 12 mM glucose. Total protein was used for normalization. (N = 3). C) Human Reconstructed Skin (HRS) generation process. HDF were seeded on bovine collagen matrix. After 21 days, Human Equivalent Dermis (HED) was produced. Human Epidermal Keratinocytes were seeded on HED. After 38 days, HRS was produced. D) Immunofluorescence analysis of MTERF3 expression in HRS epidermis grown in 5.55 mM or 12 mM glucose. Signal intensity was quantified by measuring MTERF3 fluorescence (purple color) normalized to the number of cells, as determined by the number of nuclei (DAPI staining; blue color). 10 images were acquired per HRS (N = 3). E) Similar analysis was performed in HRS dermis. F) Determination by Simple WES of the protein expression level of various mitochondrial respiratory chain subunits in human dermal fibroblasts (HDF): complex I (NADH:Ubiquinone Oxidoreductase Subunit B8), complex II (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit), complex III (Ubiquinol-Cyt C Reductase Core Protein 2), complex IV (Cyt C Oxidase Subunit 4) and complex V (ATP synthase alpha subunit) in HDF cultivated 48 h in DMEM with 5.55 mM or 12 mM of glucose. Total protein loading was used for normalization (N = 3). Similar analysis was performed in G) Human Equivalent Dermis (HED, N = 3) and H) Human Reconstructed Skin (HRS, N = 3). All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Unpaired t -test was for all panels.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Produced, Immunofluorescence, Fluorescence, Staining

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: MTERF3 ectopic overexpression represses OXPHOS. A) Proteomic study of HDF overexpressing human MTERF3 (MTERF3OE) as compared to wild-type control expressing the empty plasmid p -lenti (N = 4). B) Schematic representation of the mitochondrial proteins reduced in expression (with Adjp<0.05) as a result of the MTERF3 overexpression (MTERF3OE) in HDF. C) Ingenuity Pathway Analysis (Z-score) of the proteins significantly altered in expression, in response to MTERF3 overexpression. D) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration, non-phosphorylating respiration (oligomycin) and uncoupled respiration (CCCP) were determined in human skin fibroblasts with MTERF3 overexpression, as compared to control cells grown in 5.55 mM or 12 mM of glucose (N = 6). E) Mitochondrial morphology study of HDF overexpressing MTERF3 and control cells grown in DMEM with 5.55 mM. Mitochondrial staining was performed using 50 nM of MitoTracker Red (N = 15). Three parameters of the mitochondrial network were analyzed using Image J: particles count, tubules length and interconnections. F) Quantification of TFAM (Transcription Factor A, Mitochondrial) RNA transcript by Taqman quantitative PCR in HDF overexpressing MTERF3, as compared to plenti-control cells. Normalization of the RT-QPCR data was performed to GusB (β-glucuronidase), N = 3. G) Growth curves of HDF lenti-control and MTERF3 overexpression grown during 7 days in DMEM with 5.55 mM or 12 mM of glucose (N = 12). H) Determination by Simple WES of the protein expression level of mitochondrial respiratory chain subunits: complex I (NADH:Ubiquinone Oxidoreductase Subunit B8), complex II (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit), complex III (Ubiquinol-Cyt C Reductase Core Protein 2), complex IV (Cyt C Oxidase Subunit 4) and complex V (ATP synthase alpha subunit) in HDF control and MTERF3 overexpression cultivated 48 h in DMEM with 5.55 mM or 12 mM of glucose. Protein normalization was performed using total proteins (N = 3). All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Unpaired t -test was used for panels A,E,F and H.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: NR2F2/Coup-TFII glucose-dependent transcription factor inhibition upregulates MTERF3. A) Promoter region of the human MTERF3 gene and regulatory sites for transcription factors binding. Identification of NR2F2 binding site using Swiss Regulon (Expasy). B) Quantification of NR2F2 (Nuclear Receptor Subfamily 2 Group F Member 2) RNA transcript by taqman quantitative PCR in HDF cultivated in DMEM with 5.55 mM, 12 mM or 25 mM of glucose. Normalization of the data was performed to GusB (β-glucuronidase) (N = 3). C) MTERF3 promoter reporter activity assay using a Gaussia-luciferase (Gluc) lentiviral approach. D) Determination by bioluminescence of the MTERF3 promoter activity in HDF with lentiviral transduction of the reporter. Fibroblasts were grown 24 h in DMEM with 5.55 mM,12 mM or 25 mM of glucose, or 5.55 mM of galactose (N = 4). Data were normalized to the number of cells. E) Dose-dependent relationships between MTERF3 promoter activation and glucose concentration in the cell culture medium. F) Quantification of NR2F2 mRNA transcript by taqman quantitative PCR in HDF transfected with esiControl and esiNR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. G) MTERF3 promoter activity determination in HDF transfected with esiNR2F2 (N = 4). H) Quantification of MTERF3 mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2R2. Normalization of data to GusB (β-glucuronidase), N = 3. I) Determination by Simple WES of MTERF3 protein expression level in HDF transfected with esiNR2F2. Protein normalization was performed to total protein loading (N = 3). J) Schematic representation of lentiviral CRISPR guide RNA for the generation of NR2F2 knock-out. K) Quantification of NR2F2 mRNA transcripts by taqman quantitative PCR in stable HDF using sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. L) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration, non-phosphorylating respiration (oligo) and uncoupled respiration (CCCP) were determined in HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. M) Quantification of RNA transcripts by taqman quantitative PCR of COQ9 (coenzyme Q9), TFAM (Transcription Factor A, Mitochondrial) and MT-RNR1 (Mitochondrially Encoded 12S RRNA), in stable HDF sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of data to GusB (β-glucuronidase), N = 3. N) Mitochondrial morphology of HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Imaging was performed using 50 nM of MitoTracker Red (N = 40). O) Schematic representation of the impact of high(12 mM)-glucose medium on NR2F2, MYCN, MTERF3 and OXPHOS. P) Quantification of MTERF3 mRNA transcript by taqman quantitative PCR in HDF transfected with esiMYCN. Normalization of the data to GusB (β-glucuronidase), N = 3. Q) MTERF3 promoter activity assay in HDF transfected with esiMYCN (N = 4). R) Determination by Simple WES of the MTERF3 protein expression level in HDF transfected with esiMYCN (N = 4). All data were expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel B, D, K, L, M and N. Unpaired t -test was used for panels F, G, H, I, P, Q and R.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Inhibition, Binding Assay, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Transduction, Activation Assay, Concentration Assay, Cell Culture, Transfection, Expressing, CRISPR, Knock-Out, Imaging

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: cFOS and NR2F2 transcription factors mediate glucose-dependent repression of GDF15 in human dermis. A) Determination by Simple WES of the protein expression level of proGDF15 in wild-type HDF expressing shcontrol or shGDF15 (N = 3). B) Expression of GDF15 in the skin from human protein expression atlas from EMBL-EBI ( https://www.ebi.ac.uk ) which includes RNA-seq analyses from tissue samples of 122 human individuals, representing 32 different tissues. The results are expressed as TPM (Transcripts Per Kilobase Million). C) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF, A549 and HEPG2 (N = 3). D) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF, 786-O and SN005 cells (N = 3). E) ELISA-Based Quantification of GDF15 secretion in HDFs. HDFs were cultured under conditions of normal (5.5 mM) and high (12 mM) glucose concentrations during 48h. GDF15 levels in the culture supernatants were quantified using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions. F) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF grown in 5.55 mM, 12 mM or 25 mM glucose (N = 3). G) GDF15 promoter activity in HDF grown 24 h in DMEM with 5.55 mM,12 mM or 25 mM of glucose or 5.55 mM of galactose (N = 4). H) Dose-dependent relationship between GDF15 promoter activation and glucose concentration in the medium. I) GDF15 gene promoter sequence with identification of the binding site for the FOS transcription factor (Swiss Regulon Expasy). J) Quantification of FOS mRNA transcript by taqman quantitative PCR in HDF cultivated in 5.55 mM or 12 mM glucose (N = 3). K) Determination by Simple WES of FOS protein expression level in HDF cultivated in 5.55 mM or 12 mM glucose (N = 3). L-M) Determination by Simple WES of GDF15 protein expression level in HDF transfected with esiFOS (N = 3). N) Quantification of GDF15 mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2F2 (N = 3). O) Quantification of GDF15 mRNA transcript by taqman quantitative PCR in HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. P–S) Quantification of ATF3, ATF4, CHOP and P53 mRNA transcripts by taqman quantitative PCR in HDF expressing siCTRL and esiNR2F2 in 5.5 mM glucose or 12 mM glucose growth medium. Normalization of the data to GusB (β-glucuronidase), N = 3. T) Schematic representation of the NR2F2-MTERF3-GDF15 axis and its control on OXPHOS function in response to glucose stress. All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel C, E and N. Unpaired t -test was used for panels A, B, H, I, J, K, L and M.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Activation Assay, Concentration Assay, Sequencing, Binding Assay, Transfection, Control

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: GDF15 inhibition by hyperglycemia or shRNA alters mitochondrial biogenesis. A) Metabolomic profile of HDF grown 48H in DMEM with 5.55 mM or 12 mM + 100 nM GDF15 of glucose and HDF expressing a shGDF15 cultivated in 5.55 mM glucose (N = 3). B) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration and uncoupled respiration (CCCP) were determined in HDF grown in 5.55 mM glucose and HDF expressing shGDF15 grown in 5.55 mM glucose or 5.55 mM glucose supplemented with 100 nM gdf15. C) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration and uncoupled respiration (CCCP) were determined in HDF grown in 5.55 mM glucose and supplemented with low doses of gdf15: 20pM, 80pM, 1 nM, 10 nM and 100 nM. D) Mitochondrial respiratory chain proteins (gene loci) specifically activated at the level of chromatin accessibility by GDF15 100 nM. E-H) Quantification of mRNA transcripts by taqman quantitative PCR for NR2F2, GDF15, MTERF3 and TFAM in HDF cultivated with 5.55 mM glucose or 5.55 mM glucose supplemented with 100 nM gdf15. Normalization of the data was performed to GusB (β-glucuronidase), N = 3. I-L) Quantification of mRNA transcripts by taqman quantitative PCR for GDF15, MAPK1, MAPK3, PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha) in HDF cultivated with 5.55 mM glucose supplemented with low doses of gdf15. Normalization of the data was performed to GusB (β-glucuronidase), N = 3 M) Quantification of TFAM mRNA transcripts by taqman quantitative PCR in HDF cultivated with 5.55 mM, 12 mM or 25 mM glucose or in HDF expressing shGDF15 grown in 5.55 mM glucose. Normalization of data was performed to GusB (β-glucuronidase), N = 3. N) Quantification of PGC1α mRNA transcript by taqman quantitative PCR in HDF cultivated with 5.55 mM, 12 mM or 25 mM glucose or in HDF expressing shGDF15 grown in 5.55 mM glucose. Normalization of the data to GusB (β-glucuronidase), N = 3. O) Quantification of PGC1α mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2F2. Normalization of data to GusB (β-glucuronidase), N = 3. P,Q) Quantification of the total Coenzyme Q10 (oxidized and reduced forms) in HDF cultivated with 5.55 mM, 12 mM or 12 mM glucose medium supplemented with 100 nM gdf15. Analysis was also performed in HDF expressing shGDF15 in 5.55 mM glucose. R) Quantification of TFAM in HDF cultivated with 5.55 mM glucose medium or medium supplemented with 20pM, 100 pM and 100 nM rGDF15. S) Summary of the regulatory network linking TFAM, PGC1α, COQ9 and COQ10. All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel A, B, F, G, I-M, P–R. Unpaired t -test was used for panels E, H and O.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Inhibition, shRNA, Expressing, Real-time Polymerase Chain Reaction, Transfection

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: CYP2C9 and CYP2C19 variants cDNA and encoded amino acid changes

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques:

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: Data search results for CYP2C9 and CYP2C19 variants in the gnomAD data bank The database search results were obtained from gnomAD ( https://gnomad.broadinstitute.org/ ) and dbSNP ( https://www.ncbi.nlm.nih.gov/projects/SNP/ ) in August 2018.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques:

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Quantitative western blot analysis of newly identified variants in CYP2C9 and CYP2C19 (*P ≤ 0.05; ***P < 0.001 vs. WT). (B) Effect of MG132 treatment on the protein levels of CYP2C9 and CYP2C19. All values are mean ± S.E.M. for three separate independent assays. ANOVA was performed to compare gene expression, followed by multiple comparisons tests for individual comparisons when significant effects were detected. *P ≤ 0.05; ***P ≤ 0.001.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Western Blot, Expressing

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Relative CYP2C9 Vivid enzyme activity. Bar graphs show normalized Vivid enzymatic activity of the CYP2C9 WT and variant proteins (nanomolar fluorescent product formed per minute per milligram of total protein); error bars depict S.D. of the mean in three independent activity assays. (B) Relative CYP2C19 Vivid enzyme activity. Bar graphs show normalized Vivid enzymatic activity of the CYP2C19 WT and variant proteins (nanomolar fluorescent product formed per minute per milligram of total protein); error bars depict S.D. of the mean in three experimental samples. Enzyme activity was analyzed using one-way ANOVA, with mean comparison for each variant genotype against the WT control performed using Dunnet’s test. Corresponding P values were multiplicity adjusted and reported as such. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Activity Assay, Variant Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) CYP2C9 Vivid enzyme activity vs. protein content: correlation of recombinant protein quantity vs. Vivid enzyme activity for CYP2C9 variant allozymes. The normalized protein quantities are plotted on the horizontal axis and the normalized Vivid enzyme activities are plotted on the vertical axis. R2 = 0.693 and P = 0.0015. (B) CYP2C19 Vivid enzyme activity vs. protein content: correlation of recombinant protein quantity vs. Vivid enzyme activity for CYP2C19 variant allozymes. The normalized protein quantities are plotted on the horizontal axis and the normalized Vivid enzyme activities are plotted on the vertical axis. R2 = 0.825 and P = 0.0003. Associations between protein content and enzyme activity were evaluated using Pearson correlations, with two-sided t test P values reported. The solid line illustrates the line of identity for the association between normalized activity and protein content.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Activity Assay, Recombinant, Variant Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Michaelis-Menten curves of the enzymatic activities of the recombinant WT and 9 variant CYP2C9 allozymes (including CYP2C9*3) toward tolbutamide (each point represents the mean ± S.D. of three separate experiments). (B) Michaelis-Menten curves of the enzymatic activities of the recombinant WT and eight variant CYP2C19 proteins (including CYP2C19*3) toward (S)-mephenytoin (each point represents the mean ± S.D. of three separate experiments). Enzyme kinetics was modeled using nonlinear regression based on the Michaelis-Menten equation.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Recombinant, Variant Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: Enzyme kinetic properties of recombinant wild-type and mutant CYP2C19 proteins for (S)-mephenytoin hydroxylation and in silico functional prediction of CYP2C19 variants The kinetic parameters for (S)-mephenytoin hydroxylation of variants 518C> CT, 556C> CT, and *3 could not be determined because the amount of produced metabolite was at or below the detection limit at the lower substrate concentrations. These data represent the mean ± S.D. of three independently performed catalytic assays. Concordance is defined as agreement of the kinetic assay result with at least two of three in silico software results.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Recombinant, Mutagenesis, In Silico, Functional Assay, Produced, Kinetic Assay, Software, Activity Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Correlation analysis between enzyme activities determined by high-throughput assay vs. velocity of product formation determined by mass spectrometric assay for CYP2C9 variant allozymes. Relative velocity values of hydroxy tolbutamide formation (nanomoles of product formed per milliliter per minute) at 50 μM tolbutamide concentration are plotted on the horizontal axis and relative enzyme activity values determined by Vivid assay (nanomolars of fluorescent product formed per minute per milligram of total protein) are plotted on the vertical axis. R2 = 0.907 and P = < 0.0001. (B) Correlation analysis between enzyme activities determined by high-throughput assay vs. velocity of product formation determined by mass spectrometric assay for CYP2C19 variants. Relative velocity values of (S)-4-hydroxy mephenytoin formation (nanomoles of product formed per milliliter per minute) at 1000 μM (S)-mephenytoin concentration are plotted on the horizontal axis and relative Vivid enzyme activity values determined by Vivid assay (nanomolars of fluorescent product formed per minute per milligram of total protein) are plotted on the vertical axis. R2 = 0.898 and P ≤ 0.0001. Associations between protein content and enzyme activity were evaluated using Pearson correlations, with two-sided t test P values reported. The solid line illustrates the line of identity for the association between enzyme activities determined by high-throughput assay and intrinsic clearance determined by mass spectrometric assay.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: High Throughput Screening Assay, Variant Assay, Concentration Assay, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

doi: 10.1073/pnas.1908207116

Figure Lengend Snippet: Fig. 1. Induction of human Leydig-like cells (hLLCs) and human adrenal-like cells (hALCs) from human induced pluripotent stem cells (hiPSCs). (A) Schematic of differentiation stages from hiPSCs into human adrenal cells (hACs) or human Leydig cells (hLCs). Genes displayed in each stage represent cellular markers of that stage. EMP, early mesenchymal progenitors. (B) Schematic strategy for the induction hALCs and hLLCs from hiPSCs. Before the specification of hALCs and hLLCs, hiPSCs were first induced into EMPs. At 2 d before the EMP induction, hiPSCs were plated on Matrigel-coated plates and cultured in mTeSR medium for pro- liferation. On induction day (ID) 0, cells were cultured in STEMdiff-ACF Mesenchymal Induction (ACF-MI) medium for 4 d and MesenCult-ACF (MC-ACF) medium for 2 d to form EMPs. (Top) hALCs were induced from EMPs by the ACF-SF1 system. On ID 6, EMPs were transferred to MesenCult-ACF Attachment Substrate-coated plates and cultured in MC-ACF medium. On ID 12, EMPs were transfected with SF-1 plasmid and exposed to dibutyryl-cAMP (dbcAMP) and desert hedgehog (DHH) in MC-ACF medium for 6 d. On ID 18, cells were exposed to dbcAMP, DHH, and human CG (hCG) in MC-ACF medium for 8 d. On ID 26, hALCs formed. (Bottom) hLLCs were induced from EMPs by the COLI-SF1 system. On ID 6, EMPs were transferred to Collagen type I (COLI)-coated plates and cultured in F12/DMEM supplemented with 10% FBS (F12/FBS) for 2 d. On ID 8, cells were transfected with SF-1 plasmid and exposed to dbcAMP and DHH in F12/FBS medium for 6 d. On ID 14, cells were exposed to dbcAMP, DHH, and hCG in F12/FBS medium for 8 d. On ID 22, hLLCs formed. (C) qRT-PCR analyses of hiPSC markers (OCT4 and NANOG), primitive streak markers (MIXL1 and TBXT), and EMP markers (PDGFRA and COUP-TFII) from hiPSCs and EMPs. The low expression of OCT4, NANOG, MIXL1, and TBXT indicate that EMPs have lost pluripotency and passed the primitive streak stage. The high expression of PDGFRA and COUP-TFII indicates the appearance of EMPs. (D and F) qRT-PCR analyses of steroidogenic cell markers in hiPSCs, hALCs, and hLLCs, respectively. Both hALCs and hLLCs highly expressed SF-1. hALCs highly expressed many steroidogenic genes but not the adrenal-specific gene CYP21B and testis-specific genes HSD17B3 and CYP19A1, in comparison to hiPSCs. The insignificant up-regulation of CYP21B in hALCs might be due to its detectable expression level in both hiPSCs and hALCs that is shown in SI Appendix, Fig. S2A, suggesting gene expression in hALCs is similar to that of hACs. hLLCs highly expressed most of the steroidogenic genes except for CYP21B, suggesting their gene expression is similar to that of hLCs. (E and G) ELISAs measuring cortisol (CORT), aldosterone (ALDO), and testosterone (T) in cell supernatants of hALCs and hLLCs, respectively. hALCs produced significantly more CORT and ALDO than T, while hLLCs produced significantly more T than CORT and ALDO. NC, GFP-transfected negative control cells. The quantitative comparison of T and CORT produced by hALCs and hLLCs is shown in SI Appendix, Fig. S2 D and E. Data in C–G are presented as mean ± SD, n = 3. In D and F, P value was generated by ANOVA. Multiple comparisons were corrected by Tukey’s t test. In E and G, P value was generated by the Student’s t test. n.s., not significant at P > 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: A total of 1 μg of Steroidogenic Factor 1 (NR5A1) human-tagged ORF cloning vector (Origene, RC207577L2) was transfected into the cells using Lipofectamine 3000 and Opti-MEM according to the manufacturer’s instruction. pLenti-C-mGFP–tagged cloning vector (Origene, PS100071) was used as the GFP-transfected negative control.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Comparison, Gene Expression, Produced, Negative Control, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

doi: 10.1073/pnas.1908207116

Figure Lengend Snippet: Fig. 3. Protein expression profile and ultrastructure of hLLCs. (A) Immunocytochemistry analyses of hLC marker HSD3B2 and adult LC marker HSD17B3. Cells with positive stains were indicated by arrows. The table next to them showed that the percentages of HSD3B2- and HSD17B3-positive cells were 93.08 ± 4.21% and 57.02 ± 9.47%, respectively. (Scale bar, 100 μm.) (B) Immunofluorescent staining of HSD3B2 (green channel) indicated that the vast majority of cells differentiated toward hLLCs. Some hLLCs also expressed LHCGR (red channel, indicated by arrow), suggesting that the function of some hLLCs is regulated by hCG/LH. (Scale bar, 100 μm.) (C) Western blot analyses of SF-1, CYP11A1, HSD3B2, CYP17A1, LHCGR, STAR, and GAPDH in hiPSCs and hLLCs. The results in- dicate that, upon the overexpression of SF-1, hLLCs highly expressed CYP11A1, HSD3B2, and CYP17A1, all of which are important for T biosynthesis. The presence of LHCGR and STAR implies that hLLCs could transfer cholesterol to mitochondria under the regulation of hCG/LH signaling. Human testicular lysate was used as a positive control. (D) Western blot analyses of HSD17B3, CYP21B, and OCT4 in hiPSCs, hLLCs, and hALCs. The specific expression of HSD17B3 in hLLCs indicates the hLC properties of hLLCs. The negligible expression of CYP21B in hLLCs suggests their poor synthetic capacity for CORT and ALDO. The undetectable expression of OCT4 in hLLCs and hALCs suggests that hiPSCs stemness had been eliminated. (E–H) Transmission electron microscopy images of hLLCs showing their ultrastructure. (E–F) hLLCs possessed condensed smooth endoplasmic reticulum (SER; indicated by arrow). (G) The swirled variety of SER (WER) is also found in hLLCs. (G and H) The presence of plentiful mitochondria (M) that were close together. Note that their cristae were more lamellar-like. Moderate amounts of lipid droplets (LDs), rough endoplasmic reticulum (RER), and Golgi (G) were also found in hLLCs. N, nucleus. (Scale bars: E and G, 500 nm; F and H, 200 nm.) The full-size TEM images are provided in SI Appendix, Figs. S9–S12.

Article Snippet: A total of 1 μg of Steroidogenic Factor 1 (NR5A1) human-tagged ORF cloning vector (Origene, RC207577L2) was transfected into the cells using Lipofectamine 3000 and Opti-MEM according to the manufacturer’s instruction. pLenti-C-mGFP–tagged cloning vector (Origene, PS100071) was used as the GFP-transfected negative control.

Techniques: Expressing, Immunocytochemistry, Marker, Staining, Western Blot, Over Expression, Positive Control, Transmission Assay, Electron Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

doi: 10.1073/pnas.1908207116

Figure Lengend Snippet: Fig. 4. Steroid production in hLLCs and hALCs. (A) Steroidogenic pathway in hALCs and hLLCs leading to the production of ALDO, CORT, and T. P5, preg- nenolone; 17α-hydroxy-P5, 17α-hydroxy-pregnenolone; DHEA, dehydroepian- drosterone; P4, progesterone; 17α-Hydroxy-P4, 17α-hydroxy-progesterone; A4, androstenedione; 11-Deoxy-B, 11-deoxycorticosterone; B, corticosterone; 11-deoxy-CORT, 11-deoxycortisol. (B–E) ELISA analyses of P5, P4, DHEA, and A4 in the media of SF-1–overexpressing hALCs and hLLCs. Compared with hALCs, hLLCs produce similar amounts of P5 (B) and less P4 (C), but greater amounts of DHEA and A4 (D and E). NC, negative controls. Data are presented as mean ± SD, n ≥3. P value was generated by the Student’s t test. n.s., not significant at P > 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: A total of 1 μg of Steroidogenic Factor 1 (NR5A1) human-tagged ORF cloning vector (Origene, RC207577L2) was transfected into the cells using Lipofectamine 3000 and Opti-MEM according to the manufacturer’s instruction. pLenti-C-mGFP–tagged cloning vector (Origene, PS100071) was used as the GFP-transfected negative control.

Techniques: Enzyme-linked Immunosorbent Assay, Generated