Journal: Communications Biology

Article Title: LRIG proteins regulate lipid metabolism via BMP signaling and affect the risk of type 2 diabetes

doi: 10.1038/s42003-020-01613-w

Figure Lengend Snippet: a Wild-type (WT) and Lrig -null (TKO) MEFs expressing the BMP reporter plasmid pGL3-BRE-luciferase were treated with the indicated concentrations of BMP4 for three hours. Thereafter, the cells were lysed, and the luciferase activity was analyzed and normalized to the control. b – d Wild-type and Lrig -null MEFs were stimulated with various concentrations of BMP4 ( b ), BMP6 ( c ), or BMP9 ( d ) for one hour followed by immunocytofluorescence analysis of nuclear phospho-Smad1/5 (pSmad1/5). e , f Wild-type and Lrig1 -null MEFs ( e ) or wild-type and Lrig3 -null MEFs ( f ) were stimulated with various concentrations of BMP4 for one hour followed by nuclear pSmad1/5 analysis. g – i Western blot analyses of canonical BMP4 signaling through pSmad1/5 and noncanonical BMP signaling through phosphorylated p38 (pp38). Wild-type and Lrig -null MEFs were stimulated with the indicated concentrations of BMP4 for one hour followed by cell lysis and Western blot analysis. Uncropped blots are shown in Supplementary Fig. . g Representative Western blots showing pSmad1/5, pp38, total Smad1, total p38, and the loading control actin. h Quantification of the pSmad1/5/actin ratios. i Quantification of the pp38/actin ratios. j Gene expression levels were analyzed in wild-type (WT) and Lrig -null (TKO) MEFs via RNA sequencing (RNAseq). The apparent number of RNAseq reads for respective gene is indicated. All the values in a – f , h and i represent the means of four biological replicates that were analyzed by three experimental repeats each. j The values represent the means of four biological replicates that were analyzed once. Error bars represent the standard deviations of means from four biological replicates. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test).

Article Snippet: Cells were starved in serum-free cell culture medium for one hour and then stimulated with BMP4, recombinant human BMP6 (PeproTech Nordic, catalog # 120-06), recombinant human GDF2/BMP9 (PeproTech Nordic, catalog # 120-07), or TGF-β1 for one hour.

Techniques: Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Lysis, RNA Sequencing Assay

Journal: EMBO Molecular Medicine

Article Title: BMP-9 mediates fibroproliferation in fibrodysplasia ossificans progressiva through TGF-β signaling

doi: 10.1038/s44321-024-00174-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The concentration of BMP-9 in mouse serum was determined using a GDF2/BMP-9 ELISA Kit (LifeSpan BioSciences, LS-F3779) in accordance with the manufacturer’s instructions.

Techniques: Transgenic Assay, Recombinant, Cell Counting, Cell Cycle Assay, Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Western Blot, Reverse Transcription, Control, Software, Real-time Polymerase Chain Reaction

Journal: Communications Biology

Article Title: LRIG proteins regulate lipid metabolism via BMP signaling and affect the risk of type 2 diabetes

doi: 10.1038/s42003-020-01613-w

Figure Lengend Snippet: Wild-type (WT) and Lrig- null (TKO) MEFs were treated with an adipogenic cocktail as described in the Methods section for the indicated times. a – c Adipogenesis of wild-type and Lrig -null MEF lines. Wild-type and Lrig -null MEFs were treated with the adipogenic cocktail for nine days followed by the quantification of adipocytes via Oil Red O staining. Shown are representative images of wild-type cells with 6% covered area ( a ) and Lrig -null cells with 1.7% covered area ( b ) (scale bar, 0.6 mm) and the quantifications of percentage area coverage for the Oil Red O stained biological replicates ( n = 8 per genotype) ( c ). d – i Relative mRNA expression levels of Lrig1 ( d ), Lrig2 ( e ), Lrig3 ( f ), Cebpb ( g ), Pparg ( h ), and Ap2 ( i ). Cells were treated as in a-c. At the indicated time points after induction, the cells were lysed and analyzed by quantitative RT-PCR. Expression was normalized to the reference gene Rn18s . Shown in d-f are wild-type cells only, whereas both wild-type and Lrig -null cells are shown in g–i, as indicated, for eight biological replicates per genotype. j – l Lipidomic analyses. Lipids were extracted from wild-type and Lrig -null MEFs before or after eight days of treatment with the adipogenic cocktail. Lipid analysis was then performed by liquid chromatography coupled with tandem mass spectrometry. Each symbol represents one experimental replicate; shown are the results of three biological replicates per genotype with four experimental replicates each. j PCA score plots of all samples, labeled according to sample category. The variation explained by PC1 and PC2 was 39.6% and 33.6%, respectively. k The score plot of the OPLS-DA model built from the lipid profiles of the 8-day samples to determine the maximal variance between the wild-type and Lrig -null sample groups. l The corresponding loading plot explaining the contributions of different lipid species to the OPLS-DA model, indicating that triglycerides (TGs) (red triangles) in the wild-type samples were highly enriched compared to in the Lrig -null samples. The lipids are labeled according to lipid class (DG: diacylglycerol, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PS: phosphatidylserine, SM: sphingomyelin, TG: triacylglycerol). m , n Role of BMP for adipogenesis in vitro. Wild-type and Lrig -null MEFs were treated as in a, without (Ctrl) or with the addition of 100 ng/ml of the BMP inhibitor noggin (Noggin) ( m ), or without (Ctrl) or with the addition of 50 ng/ml of BMP4 (BMP4) ( n ). Adipogenesis was scored through Oil Red O staining as described under a . In c – i , m and n the means of the biological replicates ( c , g – i , m and n , n = 8 per genotype; d – f , n = 3) are shown by horizontal lines, and the means of the individual biological replicates analyzed by three experimental repeats are shown by dots and squares. Error bars represent the standard deviations of the means of the biological replicates. ns P > 0.05, * P < 0.05, ** P < 0.01 (Student’s t -test).

Article Snippet: Cells were starved in serum-free cell culture medium for one hour and then stimulated with BMP4, recombinant human BMP6 (PeproTech Nordic, catalog # 120-06), recombinant human GDF2/BMP9 (PeproTech Nordic, catalog # 120-07), or TGF-β1 for one hour.

Techniques: Staining, Expressing, Quantitative RT-PCR, Liquid Chromatography, Mass Spectrometry, Labeling, In Vitro

Journal: Communications Biology

Article Title: LRIG proteins regulate lipid metabolism via BMP signaling and affect the risk of type 2 diabetes

doi: 10.1038/s42003-020-01613-w

Figure Lengend Snippet: a Wild-type (WT) and Lrig -null (TKO) MEFs expressing the BMP reporter plasmid pGL3-BRE-luciferase were treated with the indicated concentrations of BMP4 for three hours. Thereafter, the cells were lysed, and the luciferase activity was analyzed and normalized to the control. b – d Wild-type and Lrig -null MEFs were stimulated with various concentrations of BMP4 ( b ), BMP6 ( c ), or BMP9 ( d ) for one hour followed by immunocytofluorescence analysis of nuclear phospho-Smad1/5 (pSmad1/5). e , f Wild-type and Lrig1 -null MEFs ( e ) or wild-type and Lrig3 -null MEFs ( f ) were stimulated with various concentrations of BMP4 for one hour followed by nuclear pSmad1/5 analysis. g – i Western blot analyses of canonical BMP4 signaling through pSmad1/5 and noncanonical BMP signaling through phosphorylated p38 (pp38). Wild-type and Lrig -null MEFs were stimulated with the indicated concentrations of BMP4 for one hour followed by cell lysis and Western blot analysis. Uncropped blots are shown in Supplementary Fig. . g Representative Western blots showing pSmad1/5, pp38, total Smad1, total p38, and the loading control actin. h Quantification of the pSmad1/5/actin ratios. i Quantification of the pp38/actin ratios. j Gene expression levels were analyzed in wild-type (WT) and Lrig -null (TKO) MEFs via RNA sequencing (RNAseq). The apparent number of RNAseq reads for respective gene is indicated. All the values in a – f , h and i represent the means of four biological replicates that were analyzed by three experimental repeats each. j The values represent the means of four biological replicates that were analyzed once. Error bars represent the standard deviations of means from four biological replicates. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test).

Article Snippet: Cells were starved in serum-free cell culture medium for one hour and then stimulated with BMP4, recombinant human BMP6 (PeproTech Nordic, catalog # 120-06), recombinant human GDF2/BMP9 (PeproTech Nordic, catalog # 120-07), or TGF-β1 for one hour.

Techniques: Expressing, Plasmid Preparation, Luciferase, Activity Assay, Control, Western Blot, Lysis, Gene Expression, RNA Sequencing

Journal: Communications Biology

Article Title: LRIG proteins regulate lipid metabolism via BMP signaling and affect the risk of type 2 diabetes

doi: 10.1038/s42003-020-01613-w

Figure Lengend Snippet: a – m Lrig -null MEFs were transduced with doxycycline-inducible LRIG1 , LRIG2 , or LRIG3 constructs or with empty vector as a noninducible control. LRIG protein expression was not induced or induced by treatment of the cells with 100 ng/ml ( a – d , m – o ) or 500 ng/ml ( e – l ) doxycycline for 24 h followed by stimulation of the cells with different concentrations of BMP4 for one hour ( a – l , n , o ) or with adipogenic cocktail for ten days ( m ). a – d Immunofluorescence analyses of nuclear pSmad1/5 in cells not induced (−dox) or induced (+dox) to express LRIG1 ( a ), LRIG2 ( b ), or LRIG3 ( c ). The empty vector served as a negative control for doxycycline treatment ( d ). e – l Western blot analyses of canonical (pSmad1/5) and noncanonical (pp38, pJnk, and pErk) BMP4 signaling. LRIG1 - or LRIG3 -inducible MEFs were induced, or not induced, with doxycycline followed by stimulation with 0 or 20 ng/ml of BMP4 for one hour. Thereafter, the cells were lysed, and the lysates were analyzed by Western blotting. e, f Representative Western blots showing pSmad1/5, total Smad1, pp38, total p38, pJnk, total Jnk, pErk, total Erk, LRIG1-FLAG ( e ), LRIG3-FLAG ( f ), and the loading control actin. Uncropped blots are shown in Supplementary Fig. . g – l Quantification of pp38 ( g , h ), pJnk ( I , j ), and pErk ( k , l ) normalized to actin in LRIG1 -inducible ( g , I , k ) or LRIG3 -inducible ( h , j , l ) MEFs. Plotted values in a – d represent means from three biological replicates, each with three experimental repeats. Error bars represent the standard deviations of means from three biological replicates. g , h Shown are four experimental repeats using an LRIG1 - or LRIG3 -inducible MEF line. Error bars show the standard deviations of the four means. i – l Shown are three experimental repeats using an LRIG1 - or LRIG3 -inducible MEF line. Error bars show standard deviations of the four means. m LRIG1 or LRIG3 expression was induced or not in Lrig -null MEFs with doxycycline followed by treatment of the cells with the adipogenic cocktail for ten days and quantification of adipocytes via Oil Red O staining. Shown are quantifications of the percentage area coverage for the Oil Red O stained biological replicates ( n = 3 per genotype and treatment). n , o LRIG1 expression was induced or not in LRIG1 -inducible HEK293T cells ( n ) and A375 cells ( o ) via the treatment of cells with doxycycline for 24 h, followed by stimulation with different concentrations of BMP4 for one hour. Immunofluorescence analyses of nuclear pSmad1/5 in HEK293T cells ( n ) or A375 cells ( o ) that were not induced (-dox) or induced (+dox) to express LRIG1 , with empty vector serving as a noninducible control. Plotted values in n and o represent the means from three independent experiments, performed as triplicates using one biological replicate of each cell line. Error bars represent standard deviations from three means. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test).

Article Snippet: Cells were starved in serum-free cell culture medium for one hour and then stimulated with BMP4, recombinant human BMP6 (PeproTech Nordic, catalog # 120-06), recombinant human GDF2/BMP9 (PeproTech Nordic, catalog # 120-07), or TGF-β1 for one hour.

Techniques: Transduction, Construct, Plasmid Preparation, Control, Expressing, Immunofluorescence, Negative Control, Western Blot, Staining

Journal: Communications Biology

Article Title: LRIG proteins regulate lipid metabolism via BMP signaling and affect the risk of type 2 diabetes

doi: 10.1038/s42003-020-01613-w

Figure Lengend Snippet: a Different LRIG protein expression levels were induced in LRIG -inducible MEFs by treating the cells with different concentrations of the inducer doxycycline. Thereafter, the cells were stimulated with 2.5 ng/ml BMP4 for one hour followed by coimmunocytofluorescence analyses of nuclear pSmad1/5 and the FLAG-epitope present as a tag on the induced LRIG proteins. Correlation plots of phosphorylated Smad1/5 versus the FLAG-LRIG protein expression levels are shown. Fitted lines indicate the linear relationship with pSmad1/5 for each LRIG protein. Pearson’s correlation coefficients for the respective genes were LRIG1, 0.9162; LRIG2, 0.6939; LRIG3, 0.9251; and empty vector, 0.1659. Shown are three experimental repeats using three biological replicates. b Lrig -null MEFs were transfected with a full-length LRIG1-GFP fusion protein (LRIG1-GFP), an LRIG1-GFP fusion protein variant lacking the cytosolic domain of LRIG1 (LRIG1-Δcyto-GFP), or empty vector (GFP) as a transfection control. Thereafter, the cells were stimulated with 20 ng/ml of BMP4 for 20 min followed by coimmunocytofluorescence analyses of nuclear pSmad1/5 and the green fluorescence from GFP fusion proteins or control GFP. The correlation plots between pSmad1/5 and GFP fluorescence are shown. The fitted lines indicate the linear relationship to pSmad1/5 for the respective construct. Pearson’s correlation coefficients for the respective constructs were as follows: full length LRIG1, 0.8943; LRIG1-Δcyto, 0.9663; GFP control, 0.2564. Shown are two experimental repeats using three biological replicates. c Lrig -null MEFs were transfected with different amounts of expression vectors encoding FLAG-tagged full-length LRIG1 (LRIG1) or FLAG-tagged LRIG1 ectodomains (LRIG1-ecto). Thereafter, the cells were stimulated with 20 ng/ml of BMP4 for 20 min followed by coimmunocytofluorescence analyses of nuclear pSmad1/5 and the FLAG-epitope. Shown are the correlation plots between pSmad1/5 and FLAG-LRIG expression levels. Fitted lines indicate the linear relationship between pSmad1/5 and the respective FLAG-LRIG construct. Pearson’s correlation coefficients for the respective constructs were as follows: full-length LRIG1, 0.7393; and LRIG1-ecto, 0.5287. Shown are two experimental repeats using three biological replicates.

Article Snippet: Cells were starved in serum-free cell culture medium for one hour and then stimulated with BMP4, recombinant human BMP6 (PeproTech Nordic, catalog # 120-06), recombinant human GDF2/BMP9 (PeproTech Nordic, catalog # 120-07), or TGF-β1 for one hour.

Techniques: Expressing, FLAG-tag, Plasmid Preparation, Transfection, Variant Assay, Control, Fluorescence, Construct