gfp targeted to mitochondria tag  (Addgene inc)

Journal: bioRxiv

Article Title: Exogenous mitochondrial transfer increases energy expenditure and attenuates adiposity gains in mice with diet-induced obesity

doi: 10.1101/2023.12.23.573206

Figure Lengend Snippet: Fluorescent donor mitochondria are taken up into inguinal and epididymal adipose tissue depots (a). Schematic of experimental design (b). Effect of mitochondrial transfer (Mito; red lines/bars/points) compared to vehicle treatment (Veh; blue lines/bars/points) on body weight (c-d), fat mass (e-f), lean mass (g-h), individual adipose depot masses (i), adipocyte size (j-k) and liver steatosis (l) and weight (m-n). Values shown are the mean ± standard error with biologically independent replicates overlayed (n=7-8 Veh and n=8-10 Mito). Data were compared by linear mixed-effects models with each individual mouse fitted as a random intercept and treatment as the main fixed variable of interest (line graphs), Student’s t-test (bar graphs, 12-week time points on line graphs, and violin plots), or analysis of covariance, with body weight and treatment as covariates (scatter plot with regression lines). Refer to each figure panel for individual p-values. Note that the adjusted R 2 value depicted in panel n reflects the proportion of variance resulting from treatment that can be explained by differences in body weight corrected for the number of variables included in the model (adjusted R 2 ; multiple R 2 = 0.866). Source data are provided in the source data file.

Article Snippet: For in vitro experiments, mitochondria were isolated from HEK293 cells expressing a GFP targeted to mitochondria tag (addgene, plasmid #50057, Watertown, MA), whereas for in vivo experiments mitochondria were isolated from liver of PhAM excised (Jackson Laboratory Stock No: 018397) or PhAMfloxed (Jackson Laboratory Stock No: 018385) crossed with Alb1-cre (Jackson Laboratory Stock No: 016832) mice using a previously described protocol .

Techniques:

Journal: Cell Death & Disease

Article Title: Obesity impairs cardiolipin-dependent mitophagy and therapeutic intercellular mitochondrial transfer ability of mesenchymal stem cells

doi: 10.1038/s41419-023-05810-3

Figure Lengend Snippet: A Schema of the development of diet-induced obesity model. The MSCs were harvested from mice fed a high-fat diet for 18 weeks (MSC-Ob) and control mice, which received a regular diet (MSC-L). The mice were subjected to measurements of body weight (b.w.), fasting glucose (FG), triglycerides (TG), and total cholesterol (TC) before sacrifice at week 24. B Flow cytometry analysis showing mitochondrial donation by GFP transduced MSCs (mito-GFP) to the vehicle (Veh), or rotenone (Rot) treated MLE-12 cells after 24 h of co-culture. C Representative images of mito-GFP-transduced MSC-L show mitochondria donation to Rot treated MLE12, which were stained with cell-tracker deep red (CTDR). Yellow arrowheads show the mito-GFP signal in MLE12 cells, and TNTs (white arrowhead) between the two cells visualized after fixing the cells and staining with phalloidin 594 (P594; blue). D The integrated density of mito-GFP signal quantified in CTDR positive MLE12 treated with Veh or Rot. E Representation of flow cytometry histograms showing mtROS levels in cell tracker green (CTG) stained MLE12 cells after co-culture with unstained MSCs ( left panel) . Histogram of the flow cytometry data with n = 5-6 from three independent experiments ( right panel) . F Immunoblot of Miro1 expression in total cell lysate and the corresponding densitometric analysis (below panel) . G The mitochondrial mass measured by flow cytometry in MSC-L and MSC-Ob after staining with mitotracker green (MTG) ( left panel) . Histogram of the flow cytometry data with n = 4 from three independent experiments ( right panel) . H Representative images of MSC-L and MSC-Ob after staining with mitotracker red (MTR) and Hoechst (blue). I Mitochondrial cell number was calculated by staining the cells with MTR and imaging analysis and presented as number of mitochondria per cell. J RT-qPCR analysis showing mtDNA content in MSC-L and MSC-Ob. K Immunoblot of PGC-1α and β-actin in cell lysate from MSC-L and MSC-Ob along with densitometric analysis ( below panel ). L Mitochondria size was calculated in images obtained after staining the cells with MTR. M , N Electron Microscopy images show imperfect cristae, more pronounced in MSC-Ob (blue arrowheads) and quantitative analysis per 100 cells. O Immunoblot and densitometric analysis of Drp1, mfn1 and β-actin. P , Q graphical representation of flow cytometry data showing mtROS in cells stained with mitoSOX and percentage depolarized mitochondria in cells stained with tetramethyl rhodamine ethyl ester (TMRE). Corresponding graphs of mitoSOX and TMRE represented as fluorescent values and percentage depolarization respectively. R Measurement of oxygen consumption rate (OCR) in MSC-L and MSC-Ob under basal and various mitochondrial complex inhibitor treatments. Data is shown as Mean±SEM with n ≥ 3. **** P < 0.001; *** P < 0.005; ** P < 0.01; * P < 0.05; ns (non-significant). Scale bars: C : 20 µm; H : 10 µm; M : 0.1 µm.

Article Snippet: MSCs were transduced with lentiviral particles having mitochondria targeting protein and GFP in downstream (Addgene) and lysosome were stained with lysotracker Deep-Red (Invitrogen, USA) followed by 3 times wash with 1X PBS.

Techniques: Control, Flow Cytometry, Co-Culture Assay, Staining, Western Blot, Expressing, Imaging, Quantitative RT-PCR, Electron Microscopy

Journal: Cell Death & Disease

Article Title: Obesity impairs cardiolipin-dependent mitophagy and therapeutic intercellular mitochondrial transfer ability of mesenchymal stem cells

doi: 10.1038/s41419-023-05810-3

Figure Lengend Snippet: A LAMP1 expression in MSCs treated with DMSO or FCCP for 2 h before the total protein lysates were prepared for immunoblotting. The right panel shows the densitometry analysis of the blots. B Representative images of LAMP1 in MSCs stained with anti-LAMP1 antibody (red) and DAPI (blue). The right panel shows the image analysis data plotted as integrated density. C MSCs were live stained with lysotracker deep-red (LTDR) and imaged in the presence of Hoechst stain (blue). The images ( C ) were quantified and represented as integrated density. D Representative images of MSC-L (D1) and MSC-Ob (D2) transduced with mitochondrial-targeted GFP (mito-GFP) and treated with DMSO or FCCP for 2 h. After fixation, the cells were stained with LAMP1 (red) and DAPI. Right panels show the line scans of the images indicating the extent of colocalization. E Mander’s coefficient showing the degree of colocalisation between LAMP1 and mitochondria. F Similarly, the images of MSC-L (F1) and MSC-Ob (F2) along with the line scans. G Image analysis ( D ) to determine the Mander’s coefficient between lysosomes and mitochondria. MSCs after transduction with mito-GFP (green) were treated with DMSO or FCCP and further stained with LTDR (red). Line scans indicate the extent of overlap between LAMP1 and mitochondria in the region selected ( F ). H , I Expression of FUNDC1, VDAC, p62 and β-actin as revealed by immunoblotting of MSCs treated with DMSO (0) or FCCP (5 µM and 10 µM). J The corresponding densitometry analysis. K LC-MS data showing the histograms of cardiolipin species detected in MSC-L and MSC-Ob cells. The cardiolipin species (with n = 3 different samples) which were significantly different are shown here while all the species detected are shown in Fig. . Mean ± SEM. **** P < 0.001; *** P < 0.005; ** P < 0.01; * P < 0.05; ns (non-significant). Scale bars: B : 50 µm; C : 100 µm; D , F : 10 µm.

Article Snippet: MSCs were transduced with lentiviral particles having mitochondria targeting protein and GFP in downstream (Addgene) and lysosome were stained with lysotracker Deep-Red (Invitrogen, USA) followed by 3 times wash with 1X PBS.

Techniques: Expressing, Western Blot, Staining, Transduction, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death & Disease

Article Title: Obesity impairs cardiolipin-dependent mitophagy and therapeutic intercellular mitochondrial transfer ability of mesenchymal stem cells

doi: 10.1038/s41419-023-05810-3

Figure Lengend Snippet: A MSCs were transduced with mito-GFP and co-cultured with MLE12 which were treated with Veh or Rot and stained with CTDR. The % GFP signal was counted in by gating MLE12 cells after 24 h. B , C Similarly, mitoSOX and TMRE staining was done in MLE12 cells after co-culture with MSCs and represented as TMRE fluorescent intensity (FI). D CTDR stained MLE12 cells were co-cultured with mito-GFP transduced MSCs and further stained with MTR. The MTR images were taken from MLE12 cells to determine mitochondrial size distribution after 48 h of co-culture. E Similar to D , with integrated density representing the mitochondrial mass in MLE12 cells by specifically counting the MTR signal in these cells. F MLE-12 cells were stained with CTDR and co-cultured with untagged MSCs for 24 h. The cells were stained with propidium iodide (PI), and flow cytometry analysis was done. MLE12 cells were gated using CTDR, and the PI signal was calculated, which is represented as % MLE12 cell death. G Immunoblots and densitometry analysis (lower panel) of Miro1 and β-actin in cell lysates prepared from various groups of MSCs. H TNT formation between co-cultured MSCs and MLE12 cells was determined by counting the number of physically attached TNTs. The data is represented as no. of TNTs per 10 2 cells. Mean ± SEM. **** P < 0.001; *** P < 0.005; ** P < 0.01; * P < 0.05; ns (non-significant).

Article Snippet: MSCs were transduced with lentiviral particles having mitochondria targeting protein and GFP in downstream (Addgene) and lysosome were stained with lysotracker Deep-Red (Invitrogen, USA) followed by 3 times wash with 1X PBS.

Techniques: Transduction, Cell Culture, Staining, Co-Culture Assay, Flow Cytometry, Western Blot

Journal: Cell Death & Disease

Article Title: Obesity impairs cardiolipin-dependent mitophagy and therapeutic intercellular mitochondrial transfer ability of mesenchymal stem cells

doi: 10.1038/s41419-023-05810-3

Figure Lengend Snippet: A Schema showing the development of HDM-induced airway allergic inflammation model. B Representative images show Mito-GFP (green) donation by MSCs to bronchial epithelial cells stained for CCSP (RED). Mito-GFP tagged MSCs were transplanted into the mice lungs via intra-tracheal infusion, and tissue sections were prepared after 24 h of infusion. C Images from B were subjected to image analysis to determine the signal of mito-GFP in CCSP positive epithelial cells and represented as integrated density. D Similarly, mitochondrial donation by MSCs was calculated using flow cytometry analysis. Single cells were prepared from lung tissue and stained for EpCAM to mark epithelial cells, which were gated to calculate the GFP signal. E , F Similarly, cells were stained with mitoSOX red and TMRE, respectively, to determine the mtROS and mitochondrial membrane potential in EpCAM stained epithelial cells. G Lung tissue was obtained from different groups of mice to prepare total lung protein (TLP). The ATP levels were measured in the TLP immediately after preparation. H Representative TUNEL images from lung tissue sections prepared from animals after 48 h of MSC transplantation. I No. of TUNEL positive cells were calculated by counting 100 bronchial epithelial cell nuclei per section, representing cell death. Mean ± SEM. **** P < 0.001; *** P < 0.005; ** P < 0.01; * P < 0.05; ns (non-significant). Scale bars: 50 µm.

Article Snippet: MSCs were transduced with lentiviral particles having mitochondria targeting protein and GFP in downstream (Addgene) and lysosome were stained with lysotracker Deep-Red (Invitrogen, USA) followed by 3 times wash with 1X PBS.

Techniques: Staining, Flow Cytometry, Membrane, TUNEL Assay, Transplantation Assay