Journal: Cell reports

Article Title: Molecular Competition in G1 Controls When Cells Simultaneously Commit to Terminally Differentiate and Exit the Cell Cycle

doi: 10.1016/j.celrep.2020.107769

Figure Lengend Snippet: (A) Cells transfected with PPARG or control siRNA were stimulated to differentiate by the standard 96 h DMI protocol. The percentage of cells in S/G2/M phases at each time point is calculated by counting the cells that expressed the APC/C reporter during the 96 h differentiation period divided by the total number of cells. The percentage of PPARG high cells was assessed by counting the cells with PPARG levels above the threshold divided by the total number of cells at the respective time point. Approximately 5,000 cells were analyzed per experiment. Representative of three biological replicates. (B) Wild-type OP9 cells transfected with PPARG or nontargeting siRNA were stimulated to differentiate with DMI. RNA samples were collected every 24 h for 144 h. Bar plots show mean ± 1 SD for three technical replicates. (C) Dual-reporter cells transfected with p21, p18, or nontargeting siRNAs were stimulated to differentiate with DMI. The number of cell divisions per cell is reported in the normalized histograms. Representative of two biological replicates. (D and E) Wild-type OP9 cells were stimulated to differentiate by addition of 1 μM rosiglitazone for 48 h. (D) p21 levels at different time points were measured by immunocytochemistry. Approximately 5,000 cells were analyzed per experiment. The values of three technical replicates (points) are plotted on top of the mean (line). (E) Chromatin immunoprecipitation (ChIP) of PPARG was performed, followed by qPCR. Three sites on the p21 promoter are shown. The promoters of insulin and Arbp/36b4 served as negative controls, and known PPARG target genes Fabp4/aP2 and Pdk4 were used as positive controls. Data are normalized to a nontargeting genomic site and IgG enrichment. Two-way ANOVA with Bonferroni’s multiple comparisons test was applied for statistical analysis. Values show mean ± SEM and are representative of two biological replicates. p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (F) Wild-type OP9 cells were transfected with p21 or control siRNA, stimulated to differentiate by addition of 1 μM rosiglitazone, and analyzed as in (A). (G) FKBPL expression under nontargeting versus PPARG knockdown were obtained from the RNA-seq data in (B). Data are reported as TPM, mean ± 1 SD. (H) Wild-type OP9 cells were transfected with FKBPL or nontargeting siRNAs and stimulated to differentiate with DMI. Stability of p21 and PPARG were assessed by adding 30 μM cycloheximide to the media 24 h after DMI addition and then fixing and staining for protein levels at different subsequent times. Approximately 5,000 cells were analyzed per experiment. Data are plotted as mean ± 1 SD of three technical replicates.

Article Snippet: The cells were incubated with primary antibodies in 2% BSA in PBS overnight at 4°C: mouse anti-PPARγ (Santa Cruz Biotech, sc-7273, 1:1,000), rabbit anti-CEBPα (Santa Cruz Biotech, sc-61, 1:1,000), mouse anti-p21 (Santa Cruz Biotech, sc-6246, 1:100), cyclinD1 (Abcam, ab137145, 1:1,000), adiponectin (Abcam, ab22554, 1:1,000), Glut4 (Santa Cruz Biotech, sc-1608, 1:500), FABP4 (R&D Systems, AF1443, 1:1,000).

Techniques: Transfection, Control, Immunocytochemistry, Chromatin Immunoprecipitation, Expressing, Knockdown, RNA Sequencing, Staining

Journal: Cell reports

Article Title: Molecular Competition in G1 Controls When Cells Simultaneously Commit to Terminally Differentiate and Exit the Cell Cycle

doi: 10.1016/j.celrep.2020.107769

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cells were incubated with primary antibodies in 2% BSA in PBS overnight at 4°C: mouse anti-PPARγ (Santa Cruz Biotech, sc-7273, 1:1,000), rabbit anti-CEBPα (Santa Cruz Biotech, sc-61, 1:1,000), mouse anti-p21 (Santa Cruz Biotech, sc-6246, 1:100), cyclinD1 (Abcam, ab137145, 1:1,000), adiponectin (Abcam, ab22554, 1:1,000), Glut4 (Santa Cruz Biotech, sc-1608, 1:500), FABP4 (R&D Systems, AF1443, 1:1,000).

Techniques: Recombinant, Staining, Activity Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Possible Involvement of Opa-Interacting Protein 5 in Adipose Proliferation and Obesity

doi: 10.1371/journal.pone.0087661

Figure Lengend Snippet: A, Images of EdU staining in the adenovirus-transfected Coxsackie-Adenovirus Receptor (CAR)-3T3-L1 adipocytes. CAR-3T3-L1 adipocytes were stained with 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by HS All-in-one Fluorescence Microscope BZ-9000. B, Number of EdU-positive CAR-3T3-L1 cells. Shown is the proportion of EdU-positive CAR-3T3-L1 cells to all nuclei. C, Images for EdU staining of CAR-3T3-L1 adipocytes analyzed by the differential interference contrast microscopy. D, CAR-3T3-L1 adipocytes co-staining with EdU and fatty acid binding protein 4 (FABP4). Images were obtained by confocal laser scanning microscopy. Oip5, Opa-interacting protein 5; βgal, β-galactosidase. Values are mean ± SE; n = 3. * P <0.05.

Article Snippet: CAR-3T3-L1 adipocytes were also stained with a rabbit anti-fatty acid binding proteins (FABP4) antibody (Catalog No. #3544, Cell signaling, Danvers, USA) as the 1 st -antibody and a goat anti-rabbit IgG conjugated Alexa 594 (Life Technologies, Gaithersburg, MD) as the 2 nd -antibody.

Techniques: Staining, Transfection, Fluorescence, Microscopy, Binding Assay, Confocal Laser Scanning Microscopy

Journal: Frontiers in Pharmacology

Article Title: Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes

doi: 10.3389/fphar.2019.01695

Figure Lengend Snippet: A greater proportion of MACS-derived adipose-derived stem cell (ASC) exhibit activity in in vitro differentiation assays compared to culture-derived ASC. (A) MACS-derived and culture-derived ASC were subjected to an in vitro adipogenic differentiation assay. The graph represents the percentage of cells which stained positive for FABP4 expression after quantification. Panel (C) shows representative images from the adipogenic differentiation assay for three donors (D1–D3) where FABP4 positive cells are stained green and nuclei are stain blue. The top row contains images of MACS-derived ASC and the bottom row contains images from culture-derived ASC. The graph in (B) represents the percentage of cells which stained positive for alizarin red after 3 weeks of culture in commercial osteogeneic differentiation media. Panel (D) shows representative images from the alizarin red assay for three donors (D1–D3). The top row contains images of MACS-derived ASC and the bottom row contains images from culture-derived ASC. * denotes a p value of < 0.05.

Article Snippet: Cells were then subjected to immunocytochemistry using a 1:200 dilution of rabbit antihuman FABP4 polyclonal antibody (Cat #10004944, Cayman Chemicals) and then incubated with a 1:200 dilution Alexa Fluor ® 488 conjugated goat antirabbit IgG secondary antibody (Cat # A11008, Molecular Probes ® ) and 1:2,000 diluted DAPI (Cat# D3571, Molecular Probes ® ).

Techniques: Derivative Assay, Activity Assay, In Vitro, Differentiation Assay, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes

doi: 10.3389/fphar.2019.01695

Figure Lengend Snippet: Ex vivo MACS-derived adipose-derived stem cell (ASC) exhibit rapid and marked changes in gene expression when subjected to standard tissue culture conditions. MACS-derived ASC were subjected to standard tissue-culture conditions and microarray analysis was performed at day 0, day 3, or day 28 post sort. (A) A heatmap was generated using a list of all transcripts which exhibited a fold change >10 when microarray data was analyzed using the Affymetrix Transcript Analysis Console software. Blue represents downregulated genes and red upregulated genes from three donor samples (1–3) at day 0, day 3, or day 28 post sort. (B) Unsupervised clustering was performed using all transcripts detected in the microarray data when processed using robust microarray average (RMA). This was then plotted as a dendogram to pictorially represent the relationship between the three donor samples (1–3) at day 0, day 3, or day 28 post sort. (C) Real-time PCR was used to validate a subset of the microarray data (target genes SCRG1, POSTN, KLF4, GREM1, C-MYC, OGN, MGP, CXCL14, FABP4, SOX2, and OCT4 as indicated) in at least four, and a maximum of eight subsequent donors (D1–D8). * denotes a p value of < 0.05, *** denotes a p value of < 0.001.

Article Snippet: Cells were then subjected to immunocytochemistry using a 1:200 dilution of rabbit antihuman FABP4 polyclonal antibody (Cat #10004944, Cayman Chemicals) and then incubated with a 1:200 dilution Alexa Fluor ® 488 conjugated goat antirabbit IgG secondary antibody (Cat # A11008, Molecular Probes ® ) and 1:2,000 diluted DAPI (Cat# D3571, Molecular Probes ® ).

Techniques: Ex Vivo, Derivative Assay, Gene Expression, Microarray, Generated, Software, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis

doi: 10.1016/j.celrep.2021.109345

Figure Lengend Snippet: (A) Metabolites that are most taken up or secreted by 3T3-L1 cells and are significantly altered after 2 days of differentiation; they were normalized to control media without cells. Data shown are from 4 biological replicates. (B) Leucine, isoleucine, and valine uptake during a time course of 3T3-L1 differentiation relative to undifferentiated state. Data shown are from 4 biological replicates. (C) Intracellular abundance of leucine, isoleucine, and valine measured from 3T3-L1 cells differentiated from 0 to 6 days and normalized to undifferentiated cells. Data shown are from 4 biological replicates. (D) Isotope abundance of 13C-leucine-derived metabolites during the time course of 3T3-L1 differentiation. Data shown are from 3 biological replicates. (E) Differentiation markers perilipin, FABP4, and PPARγ were analyzed by western blot after 8 days of differentiation in DMEM or leucine-free DMEM with or without supplementation of 10 mM dimethyl-α-ketoglutarate (α-kg). Presented replicates are biological. Data are representative of 3 independent experiments. Error bars indicate mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Article Snippet: Rabbit monoclonal FABP4 , Cell signaling , Cat# 3544T; RRID:AB_2278257.

Techniques: Control, Derivative Assay, Western Blot

Journal: Cell reports

Article Title: SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis

doi: 10.1016/j.celrep.2021.109345

Figure Lengend Snippet: (A) BCAA pathway schematic. (B) Protein expression of BCAA enzymes analyzed by western blot from 3T3-L1 cells undifferentiated or differentiated in a time course as indicated. PPARγ, c/EBPδ, and FABP4 used as markers of distinct differentiation stages. Biological duplicates presented. (C) qPCR analysis of Adipoq, Bcat2, Bckdha, and Mccc1 gene expression after a differentiation time course, as indicated. Three biological replicates are presented. (D) qPCR analysis of Adipoq, Bcat2, Bckdha, and Mccc1 gene expression after a 1-day treatment with 20 μM T0070907. Three biological replicates were used. (E) Expression of BCAA enzymes after a 1-day treatment with or without 10 μM or 20 μM T0070907, analyzed by western blot. Undifferentiated samples (0 d) were used as controls. Biological duplicates are presented. (F) Expression of BCAA enzymes after a 6-day treatment with or without 10 μM T0070907, analyzed by western blot. Undifferentiated samples (0 d) were used as controls. Biological triplicates are presented. (G) Isotope abundance of 13C-leucine-derived metabolites in 3T3-L1 cells differentiated for 1 or 5 days treated with or without 20 μM T0070907. Undifferentiated samples (day 0) were used as controls. Data shown are from 3 biological replicates. Error bars indicate mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. All data are representative of 2–3 independent experiments.

Article Snippet: Rabbit monoclonal FABP4 , Cell signaling , Cat# 3544T; RRID:AB_2278257.

Techniques: Expressing, Western Blot, Gene Expression, Derivative Assay

Journal: Cell reports

Article Title: SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis

doi: 10.1016/j.celrep.2021.109345

Figure Lengend Snippet: (A) Model for SIRT4 regulation of BCAA catabolism. (B) Relative leucine uptake from control and SIRT4-overexpressing 3T3-L1 cells differentiated from 0 to 6 days, normalized to control media without cells. Data depict biological quadruplicates. (C) Intracellular abundance of leucine from control and SIRT4-overexpressing 3T3-L1 cells differentiated from 0 to 6 days. Data depict biological quadruplicates. (D) Isotope abundance of 13C-leucine-derived metabolites in control or shSIRT4 3T3-L1 cells differentiated for 0, 2, and 4 days. Data represent biological triplicates. (E) α-Ketoisocaproic-acid-mediated oxygen consumption rate (OCR) in permeabilized control or shSIRT4 3T3-L1 cells differentiated for 0, 1, and 3 days. (F) α-Ketoisocaproic-acid-mediated OCR in permeabilized shControl or shMCCC1 3T3-L1 cells differentiated for 0 and 2 days. (G) Quantification of relative oil red O in shControl or shMCCC1 3T3-L1 cells differentiated for 8 days. Six biological replicates were used. (H) MCCC1 activity in shControl or shSIRT4 3T3-L1 cells differentiated for 2 days. Four biological replicates were used. (I) Western blot analysis of perilipin and FABP4 in 3T3-L1 cells differentiated 6 days overexpressing SIRT4 or control vector and shMCCC1 or control plasmid. Biological duplicates are presented. Error bars indicate mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. All data are representative of 2–3 independent experiments.

Article Snippet: Rabbit monoclonal FABP4 , Cell signaling , Cat# 3544T; RRID:AB_2278257.

Techniques: Control, Derivative Assay, Activity Assay, Western Blot, Plasmid Preparation

Journal: Cell reports

Article Title: SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis

doi: 10.1016/j.celrep.2021.109345

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal FABP4 , Cell signaling , Cat# 3544T; RRID:AB_2278257.

Techniques: Recombinant, Modification, shRNA, Plasmid Preparation, Software

Journal: Molecular Medicine

Article Title: Arctiin, a lignan compound, enhances adipose tissue browning and energy expenditure by activating the adenosine A 2A receptor

doi: 10.1186/s10020-025-01249-8

Figure Lengend Snippet: ARC Promotes Adipocyte Browning In Vitro. A Schematic representation of C3H10 T1/2 MSCs differentiation. B Cell viability of C3H10 T1/2 MSCs was assessed at various concentrations of Arctiin. C , D Immunoblotting and quantification of UCP1 and Fabp4 in treated C3H10 T1/2 MSCs derived adipocytes. E , F Expression of UCP1, PGC1-α, Fabp4, and PPARγ in C3H10 T1/2 cells. G Representative fluorescence images showing the expression of UCP1 (purple) and the status of lipid droplets stained with BODIPY 493/503 (green) in the treated cells after induction. Scale bar, 20 μm. H , I Oil Red O staining of differentiated adipocytes and area of lipid droplets. Scale bar, 20 μm. J , K Western blot analyses of lipolysis markers. L , M Immunofluorescence analysis of MitoTracker and quantification of mitochondrial content of cells according to ImageJ densitometry. Scale bar, 20 μm. N , O Expression and quantification of OXPHOS complexes I-V(CI-CV). Statistical analysis was done by One-way ANOVA. Control VS Rosi; Control VS ARC. All data are expressed as the means ± SD ( n = 3). * P < 0.05 , ** P < 0.01 , *** P < 0.001 , **** P < 0.0001

Article Snippet: Primary antibodies in appropriate concentration were incubated overnight as following, UCP1 (Sigma, U6382), Fabp4 (CST, 13,368), PPARγ (Santa Cruz, B-5), PGC-1α (Santa Cruz, D-5), ATGL (Proteintech, 55,190–1-AP), HSL (CST, 4107 T), phospho-HSL (CST, 4139), total OXPHOS rodent cocktail (Abcam, ab110413), A2AR (Santa Cruz, SC-32261), P-CREB (Santa Cruz, SC-81486), CREB (Santa Cruz, 377,154), P-PKA (CST, 4781), PKA (CST, 4782), Tubulin (Boster, M05613-4), GAPDH (Boster, BM3874).

Techniques: In Vitro, Western Blot, Derivative Assay, Expressing, Fluorescence, Staining, Immunofluorescence, Control

Journal: Communications Biology

Article Title: GDE7 produces cyclic phosphatidic acid in the ER lumen functioning as a lysophospholipid mediator

doi: 10.1038/s42003-023-04900-4

Figure Lengend Snippet: a Wild-type (WT), GDE7-deficient (KO) MCF-7 cells, and KO cells overexpressing mGDE7 (RE) were cultured in serum-free medium for 20 h. Total RNA was isolated and analyzed by reverse transcription qPCR for mRNA levels of CD36 , CYP27A1 , and PPARG . Values are expressed as the ratios to the GAPDH levels (mean values ± S.D., n = 4). Dunnett’s test was conducted for analysis. ** P < 0.01, *** P < 0.001 (vs. KO). b 3T3-L1 cells overexpressing hGDE7 (h7) and control 3T3-L1 cells (–) were cultured in serum-free medium with or without 1 μM rosiglitazone (ROSI) for 20 h. Total RNA was isolated and analyzed by reverse transcription qPCR for mRNA levels of Cd36 , Cyp27a1 , Gdpd3 (endogenous GDE7), GDPD3 (exogenous GDE7), Adipoq , Fabp4 , and Pparg . Values are expressed as the ratios to the Gapdh levels (mean values ± S.D., n = 3). Tukey test was conducted for analysis. * P < 0.05, ** P < 0.01, *** P < 0.001. c 3T3-L1 cells overexpressing hGDE7 (h7) and control 3T3-L1 cells (–) were treated with or without 1 μM ROSI in the presence of 10% calf serum for 1 week (medium change every other day). The cell lysates containing equal amounts of proteins were subjected to immunoblotting with anti-PPARγ, -adiponectin, -FABP4, and -CD36 antibodies. GAPDH was used as a loading control. Different blots were used for each antibody. All the experiments were repeated at least twice.

Article Snippet: Anti-PPARγ (#2435), anti-adiponectin (#2789), anti-CD36 (#28109), and anti-FABP4 (#2120) antibodies were from Cell Signaling Technology (Danvers, MA).

Techniques: Cell Culture, Isolation, Reverse Transcription, Control, Western Blot