Journal: Cell reports

Article Title: PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics.

doi: 10.1016/j.celrep.2023.112895

Figure Lengend Snippet: Figure 3. PGAM5 interacts with MFN2 and DRP1 (A) Interaction of PGAM5 WT GFP with mitochondrial dynamics regulatory proteins was analyzed by western blotting after coIP. Empty vector was used as the control. 5% of the total lysates were analyzed as input. (B) coIP analysis of PGAM5 and MFN2/DRP1 interaction. 293T cells were transfected with a plasmid encoding PGAM5 WT GFP or empty vector. At 48 h after transfection, cells were treated with either DMSO (vehicle control) or with 20 mM CCCP for 2 h. Cell lysates were immunoprecipitated with anti-GFP beads and analyzed by immunoblot (IB). 5% of the total lysates were analyzed as input. (C) MFN2 was normalized to PGAM5 WT GFP pull-down, and relative binding amounts in DMSO- and CCCP-treated conditions were plotted. The bar graph shows the comparison of the relative MFN2 binding between DMSO- and CCCP-treated conditions. Values are means ± SEM (n = 4). ***p = 0.0004 (paired t test).

Article Snippet: DRP1 K38A (addgene-37243), and MFN2WT (addgene - 28010) were originally purchased from addgene and further sub-cloned into different vectors.

Techniques: Western Blot, Plasmid Preparation, Control, Transfection, Immunoprecipitation, Binding Assay, Comparison

Journal: Cell reports

Article Title: PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics.

doi: 10.1016/j.celrep.2023.112895

Figure Lengend Snippet: Figure 4. PGAM5 functions independently of DRP1 but requires MFN2 activity (A) Confocal images of mitochondrial morphology in U2OS cells stably expressing control sh (right) and DRP1 sh (left). Confocal images of mitochondrial morphology in control sh and DRP1 sh stable U2OS cells transiently transfected with either PGAM5 H105A GFP or DMM GFP. Scale bars, 10 mm. (B) Zoomed insets of the images of (A). The region zoomed in on is marked with the white box. (C) Quantification of the mitochondrial aspect ratio of control and DRP1 knockdown cells with or without expressing PGAM5 H105A GFP or WT DMM GFP. Values are presented as mean ± SEM (n = 3) using GraphPad Prism. p values: control (non-transfected): DRP1 sh (non-transfected) **p = 0.0027; DRP1 sh (non- transfected): DRP1 sh/H105A **p = 0.0011; DRP1 sh (non-transfected): DRP1 sh/DMM ****p < 0.0001 (two-way ANOVA with Tukey’s multiple comparison test). (D) Confocal images of mitochondrial morphology in U2OS cells stably expressing control sh (right) and MFN2 sh (left). Confocal images of mitochondrial morphology in control sh and MFN2 sh stable U2OS cells transiently transfected with either PGAM5 WT or DN35 OMM GFP. Scale bars, 10 mm. (E) Zoomed insets of the images of (D). The regions zoomed in on have been marked with white boxes. (F) Quantification of the mitochondrial aspect ratio of control and MFN2 knockdown (KD) cells with or without expressing PGAM5 WT or DN35 OMM GFP. Vales are presented as mean ± SEM (n = 3) using GraphPad Prism. p values: control sh (non-transfected): MFN2 sh ***p = 0.0005; control sh (non-transfected): MFN2 sh/WT OE ***p = 0.0007; control sh (non-transfected): MFN2 sh/DN35 OMM **p = 0.0090 (two-way ANOVA with Tukey’s multiple comparison test).

Article Snippet: DRP1 K38A (addgene-37243), and MFN2WT (addgene - 28010) were originally purchased from addgene and further sub-cloned into different vectors.

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Knockdown, Comparison

Journal: Endocrinology

Article Title: The Balance of PI3K and ERK Signaling Is Dysregulated in Prolactinoma and Modulated by Dopamine

doi: 10.1210/en.2017-03135

Figure Lengend Snippet: Stable expression of constitutively active PI3K increases GH4T2 cell proliferation but does not increase colony formation. Analysis of GH4T2 protein expression, proliferation, and colony formation with expression of constitutively active PI3K is shown. GH4T2 cells were transduced with retrovirus encoding PIK3CA-E545K or empty vector as a control. Cells were maintained in complete medium with 2 μg/mL puromycin for selection. (a) Western blot analysis of GH4T2 cells for p-s6K expression. GH4T2 cells were serum starved overnight (0.05% FBS). Whole-cell extracts were separated by SDS-PAGE and probed with anti–p-s6K and anti-tubulin antibodies (n = 3; a representative experiment is shown). (b) GH4T2 cells were maintained in complete media and fresh media were added on days 2 and 4. Crystal violet staining was completed as described in “Materials and Methods” (n = 3; a representative experiment is shown). *P < 0.05 vs control. (c) Analysis of 2D cell colony formation. Cells were maintained in complete media and fresh media were added every other day (n = 3). Data are shown as means ± SEM.

Article Snippet: The pBabe puro HA PIK3CA E545K plasmid was a gift from Jean Zhao (Addgene plasmid no. 12525) ( 23 ), and pBABE-puro was a gift from Hartmut Land, Jay Morgenstern, and Bob Weinberg (Addgene plasmid no. 1764) ( 24 ).

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Selection, Western Blot, SDS Page, Staining

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