addgene pbabe bleo human pparγ2  (Addgene inc)

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: 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 biosynthesis blocks adipogenesis by downregulating the expression of key transcriptional regulators. A , Western blotting analysis of primary preadipocytes differentiated for 8 days after adding 6MP or MIZ at day 0 and then every other day, at day 2 and then every other day, or at day 4 and then every other day. Duplicate samples represent two biological replicates. B , cells were treated as in ( A ), and Oil Red O staining was performed. Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. C , MIZ and 6MP were added at the start of primary SVF differentiation. Cells were differentiated for 1, 2, or 4 days. C/EBPδ, C/EBPβ, PPARγ, C/EBPα, and tubulin were analyzed by Western blotting. Duplicate samples represent two biological replicates. D , 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 25 μM MIZ or DMSO control for 6 days. Lysates were analyzed by Western blotting as indicated. The data are representative of three independent experiments. 6MP, 6-mercaptopurine; C/EBP, CCAAT/enhancer-binding protein; DMSO, dimethyl sulfoxide; MIZ, mizoribine; PPARγ, peroxisome proliferator–activated receptor γ.

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, Expressing, Western Blot, Staining, Stable Transfection, Control, Plasmid Preparation, Binding Assay