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

Article Title: ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

doi: 10.1073/pnas.2319301121

Figure Lengend Snippet: Fig. 1. ADH1B deficiency in ASC alters lipid droplet formation and triglyceride content. Data were obtained in ASC, ASC with a CRISPR-Cas9-mediated ADH1B- knockout (KO), and ASC transduced with a Cas9/scramble gRNA plasmid corresponding to control (CTL) cells. (A) Timeline representation of the ASC differentiation process using a hormonal cocktail. IBMX: 3-isobutyl-1-methylxanthine; D0: day 0 (undifferentiated state); D10: day 10; D20: day 20. (B) ADH1B expression in ASC during adipocyte differentiation and validation of ADH1B KO in ASC at D0 and at D20. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. (C) Adipocyte differentiation assessed by Oil Red-O lipid staining. ASC preadipocytes were studied during adipocyte differentiation for 20 d. First and second lines: representative pictures of cell dishes by optical microscopy. Images are representative of three independent experiments. Third and fourth lines: representative images of fluorescence microscopy after staining of intracellular lipids (Oil Red-O, red) and nuclei (DAPI, blue). Images are representative of three independent experiments. (D) Quantification of Oil Red-O fluorescence normalized to DNA content (DAPI). Results are expressed as means ± SEM of three independent experiments. (E) Intracellular triglyceride contents were measured at D0 and D20 in ASC, CTL, and ADH1B KO cells. The measurements are representative of three independent experiments. (F) Protein expression of adipocyte markers obtained by western blotting during in vitro adipocyte differentiation of ASC cells at D0 and D20. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. PPARγ: peroxisome proliferator-activated receptor-gamma; C/EBPα: CCAAT/enhancer-binding protein-alpha; SREBP-1c: sterol regulatory element-binding protein-1c; FAS: fatty acid synthase. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. designates nonspecific bands.

Article Snippet: Fève Lab at CRSA, Paris N/A Fetal calf serum Sigma- Aldrich Cat# F7524 Newborn calf serum Biosera Cat# CA- 1151500 Chemicals, Peptides, and Recombinant Proteins 2,7- dichlorodihydrofluorescein diacetate (CM- H2DCFHDA) Sigma- Aldrich Cat# C6827 3- isobutyl- 1- methyl xanthine (IBMX) Sigma- Aldrich Cat# I7018 9- cis- retinoic acid (CAS 5300- 03- 8) Santa Cruz Biotechnology Cat# sc- 205589 All- trans- retinoic acid (CAS 302- 79- 4) Sigma- Aldrich Cat# R2625 All- trans- retinol (CAS 68- 26- 8) Sigma- Aldrich Cat# R7632 DAPI Sigma- Aldrich Cat# D1306 Dexamethasone Sigma- Aldrich Cat# D4902 Fibrobast Growth Factor- 2 (FGF- 2) PeproTech Cat# 100- 18B GlutaMAX Thermo Fisher Scientific Cat# 35050061 G- 418 Sulfate Sigma- Aldrich Cat# G418- RO HEPES Thermo Fisher Scientific Cat# 15630056 Insulin Sigma- Aldrich Cat# I0516 Oil Red- O Sigma- Aldrich Cat# O0625 Paraformaldehyde Thermo Fisher Scientific Cat# J19943.K2 Penicillin/streptomycin Thermo Fisher Scientific Cat# 11548876 Rosiglitazone Sigma- Aldrich Cat# D2408 Critical Commercial Assays Human MSC Kit buffer Lonza Cat# VPE- 1001 MycoAlertTM PLUS Mycoplasma Detection Kit Lonza Cat# LT07- 701 QuantiChromTM Alcohol dehydrogenase Assay Kit BioAssay Systems Cat# DADH- 100 QuikChange II Site- directed mutagenesis kit Agilent Technologies Cat# 200523 Triglyceride Infinity KitTM Thermo Fisher Scientific Cat# TR22421 TurboFectTM Thermo Fisher Scientific Cat# R0532 Recombinant DNA lentiCRISPR v2 Addgene Cat# 52961 pCMV6- entry- GFP Origene Cat# PS100026 pCMV- ADH1B WT- Flag Origene Cat# RC205391L3 pCMV- ADH1B c.937C>T- Flag Described in Methods N/A Software and Algorithms Multi- Experiment Viewer (MeV) WebMev N/A Prism GraphPad Software N/A Web resources CADD https://cadd.gs.washington.edu CRISPOR http://crispor.tefor.net/D ow nl oa de d fr om h ttp s: //w w w .p na s. or g by 1 03 .1 39 .1 0.

Techniques: CRISPR, Knock-Out, Transduction, Plasmid Preparation, Control, Expressing, Biomarker Discovery, Molecular Weight, Western Blot, Staining, Microscopy, Fluorescence, In Vitro, Binding Assay

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

Article Title: ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

doi: 10.1073/pnas.2319301121

Figure Lengend Snippet: Fig. 2. ADH1B deficiency can be bypassed by 9-cis RA treatment, but not by rosiglitazone. Data were obtained in ASC, ASC with a CRISPR-Cas9-mediated ADH1B- knockout (KO), and ASC transduced with a Cas9/scramble gRNA plasmid corresponding to control (CTL) cells. Adipocyte differentiation was induced in the presence or not of 9-cis RA (9-cis RA). Cells are studied at D20 postinduction. (A) Schematic representation of the link between ADH1B, 9-Cis RA, and adipocyte differentiation. The yellow square corresponds to 9-Cis RA. (B) Adipocyte differentiation assessed by Oil Red-O lipid staining. First line: representative pictures of cell dishes by optical microscopy. Second line: representative images of fluorescence microscopy after staining of intracellular lipids (Oil Red-O, red) and nuclei (DAPI, blue). Images are representative of three independent experiments. (C and G) Quantification of Oil Red-O fluorescence normalized to DNA content (DAPI) at D20. Results are expressed as means ± SEM of three independent experiments. (D) Intracellular triglyceride contents were measured at D20 in ASC, CTL, and ADH1B KO cells. The measurements are representative of three independent experiments. (E) Protein expression of adipocyte markers obtained by western blotting during in vitro adipocyte differentiation of ASC at D20. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. PPARγ: peroxisome proliferator-activated receptor-gamma; C/EBPα: CCAAT/enhancer-binding protein-alpha; SREBP-1c: sterol regulatory element-binding protein-1c; FAS: fatty acid synthase. (F) Representative images of fluorescence microscopy after staining of intracellular lipids (Oil Red-O, red) and nuclei (DAPI, blue) in ADH1B-KO ASC. The first line depicts KO cells without rosiglitazone, while the second line shows those treated with it. (G) Quantification of Oil Red-O fluorescence normalized to DNA content (DAPI) at D20. Results are expressed as means ± SEM of three independent experiments. **P < 0.01, ****P < 0.0001, n.s.: nonsignificant.

Article Snippet: Fève Lab at CRSA, Paris N/A Fetal calf serum Sigma- Aldrich Cat# F7524 Newborn calf serum Biosera Cat# CA- 1151500 Chemicals, Peptides, and Recombinant Proteins 2,7- dichlorodihydrofluorescein diacetate (CM- H2DCFHDA) Sigma- Aldrich Cat# C6827 3- isobutyl- 1- methyl xanthine (IBMX) Sigma- Aldrich Cat# I7018 9- cis- retinoic acid (CAS 5300- 03- 8) Santa Cruz Biotechnology Cat# sc- 205589 All- trans- retinoic acid (CAS 302- 79- 4) Sigma- Aldrich Cat# R2625 All- trans- retinol (CAS 68- 26- 8) Sigma- Aldrich Cat# R7632 DAPI Sigma- Aldrich Cat# D1306 Dexamethasone Sigma- Aldrich Cat# D4902 Fibrobast Growth Factor- 2 (FGF- 2) PeproTech Cat# 100- 18B GlutaMAX Thermo Fisher Scientific Cat# 35050061 G- 418 Sulfate Sigma- Aldrich Cat# G418- RO HEPES Thermo Fisher Scientific Cat# 15630056 Insulin Sigma- Aldrich Cat# I0516 Oil Red- O Sigma- Aldrich Cat# O0625 Paraformaldehyde Thermo Fisher Scientific Cat# J19943.K2 Penicillin/streptomycin Thermo Fisher Scientific Cat# 11548876 Rosiglitazone Sigma- Aldrich Cat# D2408 Critical Commercial Assays Human MSC Kit buffer Lonza Cat# VPE- 1001 MycoAlertTM PLUS Mycoplasma Detection Kit Lonza Cat# LT07- 701 QuantiChromTM Alcohol dehydrogenase Assay Kit BioAssay Systems Cat# DADH- 100 QuikChange II Site- directed mutagenesis kit Agilent Technologies Cat# 200523 Triglyceride Infinity KitTM Thermo Fisher Scientific Cat# TR22421 TurboFectTM Thermo Fisher Scientific Cat# R0532 Recombinant DNA lentiCRISPR v2 Addgene Cat# 52961 pCMV6- entry- GFP Origene Cat# PS100026 pCMV- ADH1B WT- Flag Origene Cat# RC205391L3 pCMV- ADH1B c.937C>T- Flag Described in Methods N/A Software and Algorithms Multi- Experiment Viewer (MeV) WebMev N/A Prism GraphPad Software N/A Web resources CADD https://cadd.gs.washington.edu CRISPOR http://crispor.tefor.net/D ow nl oa de d fr om h ttp s: //w w w .p na s. or g by 1 03 .1 39 .1 0.

Techniques: CRISPR, Knock-Out, Transduction, Plasmid Preparation, Control, Staining, Microscopy, Fluorescence, Expressing, Western Blot, In Vitro, Molecular Weight, Binding Assay

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

Article Title: ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

doi: 10.1073/pnas.2319301121

Figure Lengend Snippet: Fig. 3. The block in adipogenesis is not rescued by retinol or small doses of ATRA. Data were obtained in ASC, ASC with a CRISPR-Cas9-mediated ADH1B-knockout (KO), and ASC transduced with a Cas9/scramble gRNA plasmid corresponding to control (CTL) cells. Adipocyte differentiation was induced in the presence or not of 9-cis RA, all-trans retinol (ATRetinol) and all-trans RA (ATRA). Cells are studied at D20 postinduction. (A and E) Adipocyte differentiation assessed by Oil Red-O lipid staining. Representative images of fluorescence microscopy after staining of intracellular lipids (Oil Red-O, red) and nuclei (DAPI, blue) are depicted. Images are representative of three independent experiments. (B and F) Quantification of Oil Red-O fluorescence normalized to DNA content (DAPI) at D20. Results are expressed as means ± SEM of three independent experiments. (C and G) Intracellular triglyceride contents were measured at D20 in ASC, CTL, and ADH1B KO cells. The measurements are representative of three independent experiments. (D) Protein expression of adipocyte markers obtained by western blotting during in vitro adipocyte differentiation of ASC at D20. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. PPARγ: peroxisome proliferator-activated receptor-gamma; C/EBPα: CCAAT/enhancer-binding protein-alpha; SREBP-1c: sterol regulatory element-binding protein-1c; FAS: fatty acid synthase. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Fève Lab at CRSA, Paris N/A Fetal calf serum Sigma- Aldrich Cat# F7524 Newborn calf serum Biosera Cat# CA- 1151500 Chemicals, Peptides, and Recombinant Proteins 2,7- dichlorodihydrofluorescein diacetate (CM- H2DCFHDA) Sigma- Aldrich Cat# C6827 3- isobutyl- 1- methyl xanthine (IBMX) Sigma- Aldrich Cat# I7018 9- cis- retinoic acid (CAS 5300- 03- 8) Santa Cruz Biotechnology Cat# sc- 205589 All- trans- retinoic acid (CAS 302- 79- 4) Sigma- Aldrich Cat# R2625 All- trans- retinol (CAS 68- 26- 8) Sigma- Aldrich Cat# R7632 DAPI Sigma- Aldrich Cat# D1306 Dexamethasone Sigma- Aldrich Cat# D4902 Fibrobast Growth Factor- 2 (FGF- 2) PeproTech Cat# 100- 18B GlutaMAX Thermo Fisher Scientific Cat# 35050061 G- 418 Sulfate Sigma- Aldrich Cat# G418- RO HEPES Thermo Fisher Scientific Cat# 15630056 Insulin Sigma- Aldrich Cat# I0516 Oil Red- O Sigma- Aldrich Cat# O0625 Paraformaldehyde Thermo Fisher Scientific Cat# J19943.K2 Penicillin/streptomycin Thermo Fisher Scientific Cat# 11548876 Rosiglitazone Sigma- Aldrich Cat# D2408 Critical Commercial Assays Human MSC Kit buffer Lonza Cat# VPE- 1001 MycoAlertTM PLUS Mycoplasma Detection Kit Lonza Cat# LT07- 701 QuantiChromTM Alcohol dehydrogenase Assay Kit BioAssay Systems Cat# DADH- 100 QuikChange II Site- directed mutagenesis kit Agilent Technologies Cat# 200523 Triglyceride Infinity KitTM Thermo Fisher Scientific Cat# TR22421 TurboFectTM Thermo Fisher Scientific Cat# R0532 Recombinant DNA lentiCRISPR v2 Addgene Cat# 52961 pCMV6- entry- GFP Origene Cat# PS100026 pCMV- ADH1B WT- Flag Origene Cat# RC205391L3 pCMV- ADH1B c.937C>T- Flag Described in Methods N/A Software and Algorithms Multi- Experiment Viewer (MeV) WebMev N/A Prism GraphPad Software N/A Web resources CADD https://cadd.gs.washington.edu CRISPOR http://crispor.tefor.net/D ow nl oa de d fr om h ttp s: //w w w .p na s. or g by 1 03 .1 39 .1 0.

Techniques: Blocking Assay, CRISPR, Knock-Out, Transduction, Plasmid Preparation, Control, Staining, Fluorescence, Microscopy, Expressing, Western Blot, In Vitro, Molecular Weight, Binding Assay

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

Article Title: ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

doi: 10.1073/pnas.2319301121

Figure Lengend Snippet: Fig. 4. Identification of an ADH1B homozygous variant affecting ADH1B homodimerization and enzyme activity in a patient with generalized lipoatrophy. (A) Genealogical tree and segregation analysis for the ADH1B variant in the family investigated herein. The arrow indicates the proband. +, normal allele; M, mutant allele. (B) Top panel: schematic representation of ADH1B transcript sequence (NM_000668.6) displaying the location of the variant identified. Bottom panel: schematic representation of ADH1B protein sequence comprising 375 amino acids. The prediction of protein domain organization was based on the UniProt database (protein reference: P00325). (C) Classification of amino acids in order of increasing hydrophobicity using a Fauchère and Pliska plot and showing the major change induced by the p.Arg313Cys variant. (D) Prediction of the effect of p.Arg313Cys variant on ADH1B 3D structure. This was performed using the MIZTLI software (https://miztli.biokerden.eu/) and the 1u3u ADH1B structure from Protein Data Bank (https://www.wwpdb.org/). Arg313 was replaced by a cysteine residue using FASPR (23). Dashed lines represent noncovalent hydrogen bonds. The p.Arg313Cys variant induces the loss of a number noncovalent interactions with other ADH1B residues, which is predicted to affect the protein stability. (E) Model of the 3D structure of ADH1B extracted from SWISS MODEL repository and corresponding to the crystal structure of a human homodimer of ADH1B (https://swissmodel.expasy.org—protein reference: P00325). The location of the p.Arg313Cys variant identified in patient 1 is indicated by a star. (F) Transfection of HEK 293 cells with plasmids encoding wild-type (WT) ADH1B and the p.Arg313Cys mutated form of the protein with a C-terminal Flag tag. Cellular extracts were subjected to native-PAGE (Upper panel) vs. SDS-PAGE (Middle panel) to evaluate the impact of the variant on ADH1B homodimerization. *: The lowest band on the gel corresponds to the one obtained with the anti-Flag antibody, since the same membrane was blotted to reveal tubulin. (G) Assessment of ADH activity in HEK 293 cells stably expressing either WT or mutant forms of ADH1B. The measurements are representative of three independent experiments. (H) WB analysis performed on ADH1B KO cells nucleofected with plasmids encoding ADH1B WT, mutant, or green fluorescent protein (GFP). (I) Optical microscopy images captured from ADH1B KO cells complemented with ADH1B WT, mutant, or GFP at D15 after adipocyte differentiation induction. (J) Measurement of intracellular triglyceride levels at D15 in ADH1B KO cells complemented with ADH1B WT, mutant, or GFP as a negative control. The measurements are representative of two independent experiments in duplicates. *P < 0.05, **P < 0.01.

Article Snippet: Fève Lab at CRSA, Paris N/A Fetal calf serum Sigma- Aldrich Cat# F7524 Newborn calf serum Biosera Cat# CA- 1151500 Chemicals, Peptides, and Recombinant Proteins 2,7- dichlorodihydrofluorescein diacetate (CM- H2DCFHDA) Sigma- Aldrich Cat# C6827 3- isobutyl- 1- methyl xanthine (IBMX) Sigma- Aldrich Cat# I7018 9- cis- retinoic acid (CAS 5300- 03- 8) Santa Cruz Biotechnology Cat# sc- 205589 All- trans- retinoic acid (CAS 302- 79- 4) Sigma- Aldrich Cat# R2625 All- trans- retinol (CAS 68- 26- 8) Sigma- Aldrich Cat# R7632 DAPI Sigma- Aldrich Cat# D1306 Dexamethasone Sigma- Aldrich Cat# D4902 Fibrobast Growth Factor- 2 (FGF- 2) PeproTech Cat# 100- 18B GlutaMAX Thermo Fisher Scientific Cat# 35050061 G- 418 Sulfate Sigma- Aldrich Cat# G418- RO HEPES Thermo Fisher Scientific Cat# 15630056 Insulin Sigma- Aldrich Cat# I0516 Oil Red- O Sigma- Aldrich Cat# O0625 Paraformaldehyde Thermo Fisher Scientific Cat# J19943.K2 Penicillin/streptomycin Thermo Fisher Scientific Cat# 11548876 Rosiglitazone Sigma- Aldrich Cat# D2408 Critical Commercial Assays Human MSC Kit buffer Lonza Cat# VPE- 1001 MycoAlertTM PLUS Mycoplasma Detection Kit Lonza Cat# LT07- 701 QuantiChromTM Alcohol dehydrogenase Assay Kit BioAssay Systems Cat# DADH- 100 QuikChange II Site- directed mutagenesis kit Agilent Technologies Cat# 200523 Triglyceride Infinity KitTM Thermo Fisher Scientific Cat# TR22421 TurboFectTM Thermo Fisher Scientific Cat# R0532 Recombinant DNA lentiCRISPR v2 Addgene Cat# 52961 pCMV6- entry- GFP Origene Cat# PS100026 pCMV- ADH1B WT- Flag Origene Cat# RC205391L3 pCMV- ADH1B c.937C>T- Flag Described in Methods N/A Software and Algorithms Multi- Experiment Viewer (MeV) WebMev N/A Prism GraphPad Software N/A Web resources CADD https://cadd.gs.washington.edu CRISPOR http://crispor.tefor.net/D ow nl oa de d fr om h ttp s: //w w w .p na s. or g by 1 03 .1 39 .1 0.

Techniques: Variant Assay, Activity Assay, Mutagenesis, Sequencing, Software, Residue, Transfection, FLAG-tag, Clear Native PAGE, SDS Page, Membrane, Stable Transfection, Expressing, Microscopy, Negative Control