Journal: Journal of lipid research

Article Title: Disruption of nucleotide biosynthesis reprograms mitochondrial metabolism to inhibit adipogenesis.

doi: 10.1016/j.jlr.2024.100641

Figure Lengend Snippet: Fig. 4. PPARγ overexpression rescues mitochondrial function induced by the loss of de novo nucleotide biosynthesis. A: OXPHOS protein levels were measured in primary SVF cells that were differentiated and treated with PPARγ inhibitor SR 16832 (2 μM or 10 μM) for 6 days (B) 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM 5FU or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. C: 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM MIZ or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. D: Live cell imaging with MTG and TMRE staining was used to assess changes in mito- chondrial membrane potential relative to mitochondrial mass in the presence or absence of 25 μM MIZ. E: ImageJ was used to measure the ratio of MTG and TMRE. All experiments were repeated two or three times with two or three biological replicates. Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ****P < 0.0001.

Article Snippet: PPARγ2 plasmid (Addgene #8859) or pBabe control was transfected into PLAT-E cells.

Techniques: Over Expression, Stable Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Live Cell Imaging, Staining, Membrane

Journal: Development (Cambridge, England)

Article Title: Nelfb promotes dermal white adipose tissue formation through RNA polymerase II-mediated adipogenic gene regulation

doi: 10.1242/dev.204976

Figure Lengend Snippet: Exogenous expression of Pparg restores adipocyte differentiation in Nelfb deleted cells. (A,B) Oil Red O staining of control cells transduced with pBABE retrovirus (CTL pBABE), Nelfb −/− cells transduced with pBABE retrovirus ( Nelfb −/− pBABE) and Nelfb −/− cells transduced with Pparg retrovirus ( Nelfb −/− Pparg). Cells were cultured in adipocyte differentiation medium for 14 days. Images were taken at 20× (A) and 60× (B) magnification ( N =3). (C,D) Staining of CTL pBABE, Nelfb −/− pBABE and Nelfb −/− Pparg cells with antibodies against perilipin 1 (Plin1; mature adipocyte marker: green) and Hoechst 33342 (nuclei: blue) at 20× (C) and 60× (D) magnification ( N =3). (E) Percent of Plin1 + cells quantified from D. (F,G) Staining of CTL pBABE, Nelfb −/− pBABE and Nelfb −/− Pparg with antibodies against Pparg (green) and Hoechst 33342 (blue) at 20× (F) and 60× (G) magnification ( N =3). (H) Percent of Pparg + cells quantified from G. (I) RT-qPCR of adipogenic genes on CTL pBABE, Nelfb −/− pBABE and Nelfb −/− Pparg cells ( N =4). Results were normalized to housekeeping gene, Gapdh . Data are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (RM one-way ANOVA followed by Tukey's multiple comparison tests for E and H; multiple unpaired t-tests for I). ns, not significant. Scale bars: 150 μm (A,C,F); 50 μm (B,D,G).

Article Snippet: The pBABE puro PPARγ2 plasmid, a gift from Bruce Spiegelman ( ; Addgene plasmid #8859 ), was used for Pparg overexpression, while the empty pBABE puro plasmid ( Addgene plasmid #110349 ) served as a negative control.

Techniques: Expressing, Staining, Control, Transduction, Cell Culture, Marker, Quantitative RT-PCR, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of nucleotide biosynthesis disrupts lipid accumulation and adipogenesis

doi: 10.1016/j.jbc.2023.104635

Figure Lengend Snippet: Inhibition of purine and pyrimidine metabolism disrupts lipid droplets in differentiating adipocytes. A , schematic depicting de novo purine synthesis and salvage pathways. Inhibitors of purine synthesis pathways in blue . B , protein levels of perilipin, FABP4, and α-tubulin in undifferentiated SVF preadipocytes or after 6 days of differentiation in the presence or the absence of 50 μM 6-mercaptopurine (6MP), 2 μM lometrexol (LOM), 25 μM mizoribine (MIZ), 10 μM leflunomide (LEF), 1 μM brequinar (BRQ), or 1 μM 5-fluororacil (5FU). Media were changed every 2 days, and drug was replenished. Data shown are from two biological replicates. C , representative images of BODIPY 493/503 staining of untreated primary SVF cells or treated with indicated drugs, differentiated for 6 days. D , quantification of Oil Red O extracted from primary adipocytes treated with indicated drugs normalized to untreated cells. All cells were differentiated for 6 days. Data shown are from three biological replicates. Statistical significance was determined using one-way ANOVA multiple comparisons test. E , protein levels of perilipin, FABP4, and α-tubulin after 6 days of differentiation of 3T3-L1 adipocytes treated as in ( B ). Data shown are from two biological replicates. F , quantification of Oil Red O staining in 3T3-L1 adipocytes treated with indicated drugs normalized to untreated cells. All cells differentiated for 6 days. Data shown are from three biological replicates. Statistical significance was determined using one-way ANOVA multiple comparisons test. G , representative images of BODIPY 493/503 staining of untreated 3T3-L1 cells or treated with indicated drugs, differentiated for 6 days. H , Western blot analysis of perilipin, FABP4, IMPDH1, and tubulin from 3T3-L1 cells infected with shControl or shIMPDH1 (two distinct shRNAs) and differentiated for 8 days. Data shown are from two biological replicates. I , representative images of BODIPY 493/503 staining of 3T3-L1 cells infected with shControl or shIMPDH1 and differentiated for 8 days. Error bars indicate mean ± SD, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. All data are representative of 2 to 3 independent experiments. FABP4, fatty acid binding protein 4; IMPDH, inosine monophosphate dehydrogenase.

Article Snippet: pBABE puro PPARγ2 plasmid was obtained from Addgene (#8859). shRNAs against IMPDH1 and DHODH were subcloned in the pLKO.1 puro vector (Addgene Plasmid #8453) at EcoRI and AgeI sites.

Techniques: Inhibition, Staining, Western Blot, Infection, Binding Assay