Journal: Nature Cell Biology

Article Title: Functional multi-organelle units control inflammatory lipid metabolism of macrophages

doi: 10.1038/s41556-024-01457-0

Figure Lengend Snippet: a Quantification of organellar volume upon LPS/IFN-γ treatment. Data represent N = 59 (0 h), N = 48 (1 h), N = 56 (6 h), N = 46 (16 h), N = 59 (24 h) cells from n = 3 (16 h, LD 0–6 h) and n = 4 (non-LD 0–6, 24 h) independent experiments. P values were calculated using one-way ANOVA with Dunnett’s post-hoc test. b Cell size of naïve and 24h LPS/IFN-γ-treated BMDMs representing n = 6 independent experiments. P value was obtained by two-tailed, unpaired t -test. c-e Images ( c ) and quantification ( d , average mitochondrial volume/cell; e , mitochondrial sphericity) showing dynamics in mitochondrial morphology of LPS/IFN-γ-activated BMDMs. Images are single z-planes and representative of n = 3 independent experiments. Scale bars: 5 µm, 1 µm (magnification) ( c ). Data represent ( d ) N = 51 (0 h), N = 76 (1 h), N = 86 (6 h) and N = 80 (24 h) and ( e ) N = 55 (0 h), N = 78 (1 h), N = 71 (6 h), N = 79 (24 h) cells from n = 3 independent experiments. P values were calculated using one-way ANOVA with Tukey’s post-hoc test. f-g Western blot analysis ( f ) and quantification ( g ) showing protein levels and activation state of DRP1 upon LPS/IFN-γ treatment. Data represent n = 3 biological replicates. P values were calculated using one-way ANOVA with Sidak’s post-hoc test ( g ). h,i Images ( h ) and quantification ( i ) showing the localization of DRP1 to mitochondria (HSP60) in LPS/IFN-γ-activated BMDMs. Scale bars: 5 µm, 1 µm (magnification) ( h ). Data represent N = 26 (0 h), N = 32 (1 h), N = 30 (6 h), N = 30 (24 h) cells from n = 3 biological replicates. P values were calculated using one-way ANOVA with Tukey’s post-hoc test ( i ). j-m WB analysis ( j,l ) and quantification ( k,m ) showing protein levels of MFN2 ( j-k ) and OPA1 ( l-m ) upon LPS/IFN-γ treatment. Data represent n = 3 biological replicates. P values were calculated using one-way ANOVA with Sidak’s post-hoc test ( k,m ). Boxes represent 25 th to 75 th percentiles, whiskers 10 th and 90 th percentiles. Dots are outliers, median is shown as line ( a,d,e,i ). The bars show the mean ± SEM ( b ) or the mean ± SD ( g,k,m ). Numerical P values are indicated in Supplementary Information Table (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, not significant (ns) P > 0.05). Source numerical data and unprocessed blots are available in source data.

Article Snippet: The following original sequences and target vectors were used: ERmoxGFP (Addgene no. 68072) and pBMN backbone (Addgene no. 1734), ERmoxVenus, mOrange2Peroxisomes2 (Addgene no. 54596), Abcd1 (Dharmacon no. MMM1013-202798649), and MigR1-IRES-GFP backbone (Addgene no. 27490), Miga2 (Dharmacon no. MMM1013-202770174) and MigR1-IRES-BFP plasmid, mApple-M1-Spastin (Addgene no. 134461) and pBMN plasmid, human Mfn2 (Addgene no. 121997) and Rmdn3 (Addgene no. 170536) and MigR1-IRES-BFP backbone.

Techniques: Two Tailed Test, Western Blot, Activation Assay

Journal: Nature Cell Biology

Article Title: Functional multi-organelle units control inflammatory lipid metabolism of macrophages

doi: 10.1038/s41556-024-01457-0

Figure Lengend Snippet: a Images depicting M-ER-P-LD units in LPS/IFN-γ-activated BMDMs (24 h) expressing BFP, MIGA2 or MIGA2 DFFAT . Images represent maximum intensity projections of n = 3 biolgocial replicates. Scale bars: 5 µm, 1 µm (magnification). b,c Pair-wise M-ER and M-LD high proximity sites ( b ) and MIGA2-regulated LD-units ( c ) in LPS/IFN-γ-treated (24 h) BMDMs expressing BFP, MIGA2 or MIGA2 DFFAT . Data represent N = 48 (BFP), N = 48 (MIGA2) and N = 40 (MIGA2 DFFAT ) cells from n = 3 biologically independent replicates. Boxes represent 25 th to 75 th percentiles, whiskers 10 th and 90 th percentiles. Dots are outliers, the median is shown as a line. P values were obtained using one-way ANOVA with Dunnett’s post-hoc test. d Western Blot analysis showing knock-down (KD) efficiency of Mfn2 or Rmdn3 in BMDMs representative of n = 1 experiment. e Pairwise ER-mitochondria HPS analysis in Mfn2 KD , Rmdn3 KD or control BMDMs. Data represent N = 32;24;29 (shCtrl; Mfn2 KD; Rmdn3 KD) cells from n = 3 biological replicates. Boxes represent 25 th to 75 th percentiles, whiskers 10 th and 90 th percentiles. Dots are outliers, the median is shown as a line. P values were calculated using one-way ANOVA with Dunnett’s post hoc test. f-h PGE 2 production of LPS/IFN-γ-treated BMDMs (24 h) upon Mfn2 and Rmdn3 KD ( f ), MFN2 and RMDN3 overexpression ( g ) or DGAT2 inhibition (DGAT2i) ( h ). Data represent n = 4 ( f , Rmdn3 KD) and n = 3 ( f , Mfn2 KD; g-h ) biologically independent experiments. P values were calculated using two-tailed, one-sample t tests ( f,h ) or one-way ANOVA with Dunnett’s post-hoc test ( g ). Bars show mean ± SEM ( f-h ). Numerical P values are available in Supplementary Information Table (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, not significant (ns) P > 0.05). Source numerical data and unprocessed blots are available in source data.

Article Snippet: The following original sequences and target vectors were used: ERmoxGFP (Addgene no. 68072) and pBMN backbone (Addgene no. 1734), ERmoxVenus, mOrange2Peroxisomes2 (Addgene no. 54596), Abcd1 (Dharmacon no. MMM1013-202798649), and MigR1-IRES-GFP backbone (Addgene no. 27490), Miga2 (Dharmacon no. MMM1013-202770174) and MigR1-IRES-BFP plasmid, mApple-M1-Spastin (Addgene no. 134461) and pBMN plasmid, human Mfn2 (Addgene no. 121997) and Rmdn3 (Addgene no. 170536) and MigR1-IRES-BFP backbone.

Techniques: Expressing, Western Blot, Knockdown, Control, Over Expression, Inhibition, Two Tailed Test

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: High glucose induces ER stress and upregulates intracellular and mitochondrial Ca 2+ levels. (a) Primary atrial myocytes under normal glucose (NG) or high glucose (HG) with or without 4-PBA for 24 hours. Quantification of GRP78 and CHOP mRNA expression. (b) Primary atrial myocytes under NG or HG with or without 4-PBA for 48 hours. GRP78 and CHOP protein level in atrial myocytes detected by Western blot analysis. (c) Quantification of GRP78 and CHOP protein level in (b). (d) Representative confocal microscopy images of fluorescence staining for Fluo-4 AM and DAPI. Scale bar, 20 μ m. (e) Quantification of Fluo-4 AM fluorescence intensity in (d). (f) Representative confocal microscopy images of fluorescence staining for Rhod2 AM and MitoTracker Green. Scale bar, 20 μ m. (g) Quantification of Rhod2 AM fluorescence intensity in (f). (h) MAM proteins such as IP 3 R1, GRP75, VDAC1, and Mfn2 protein level in atrial myocytes detected by Western blot analysis. (i) Quantification of IP3R1, GRP75, VDAC1, and Mfn2 protein level in (h). Data are mean ± SEM, n = 4 independent experiments, ∗ p < 0.05, # p < 0.01, ANOVA with Bonferroni posttest.

Article Snippet: HL-1 cells were seeded into six-well plates, when cells reached 70-80% confluence, they were transfected with control siRNA (sc-37007) or Mfn2 siRNA (sc-60077, both Santa Cruz Biotechnology, CA) by using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA) for 24 h. Then, HL-1 cells were treated with 200 ng/mL TM (Solarbio Life Sciences, Beijing) for the next 24 h. DS16570511 (MedChemExpress, USA) is a cell-permeable inhibitor of the mitochondrial calcium uniporter, which blocks the MCU-dependent increase of Ca 2+ influx.

Techniques: Expressing, Western Blot, Confocal Microscopy, Fluorescence, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: Mfn2 siRNA disrupts endoplasmic reticulum- (ER-) mitochondria tethering in tunicamycin- (TM-) injured HL-1 cells. (a) Changes in GRP78 and Mfn2 levels induced by TM were examined using Western blot analysis. The silencing efficiency of Mfn siRNA also shown in (a). (b) Quantification of GRP78 and Mfn2 protein level in (a). (c) Quantification of Mfn2 mRNA expression. (d) Ultrastructural changes in HL-1 cells (green stars depicting the mitochondria, white arrowheads depicting the endoplasmic reticulum). Transmission electron micrograph images (red arrowheads depicting the ER-mitochondria contacts) and measurements of the ER-mitochondria distance (e). Scale bar, top 1 μ m, middle 2 μ m, and bottom 500 nm. Data are mean ± SEM, n = 4 independent experiments, # p < 0.01, ANOVA with Bonferroni posttest.

Article Snippet: HL-1 cells were seeded into six-well plates, when cells reached 70-80% confluence, they were transfected with control siRNA (sc-37007) or Mfn2 siRNA (sc-60077, both Santa Cruz Biotechnology, CA) by using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA) for 24 h. Then, HL-1 cells were treated with 200 ng/mL TM (Solarbio Life Sciences, Beijing) for the next 24 h. DS16570511 (MedChemExpress, USA) is a cell-permeable inhibitor of the mitochondrial calcium uniporter, which blocks the MCU-dependent increase of Ca 2+ influx.

Techniques: Western Blot, Expressing, Transmission Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: Mfn2 siRNA inhibited mitochondrial Ca 2+ overload during ER stress induced by TM. (a) Representative confocal microscopy images of fluorescence staining for Fluo-4 AM and DAPI. Scale bar, 20 μ m. (b) Quantification of Fluo-4 AM fluorescence intensity in (a). (c) Representative confocal microscopy images of fluorescence staining for Rhod2 AM and MitoTracker Green. Scale bar, 20 μ m. (d) Quantification of Rhod2 AM fluorescence intensity in (c). (e) Mitochondria Ca 2+ proteins such as VDAC1 and MCU protein level in atrial myocytes detected by Western blot analysis. (f) Quantification of VDAC1 and MCU protein level in (e). Data are mean ± SEM, n = 4 independent experiments, # p < 0.01, ANOVA with Bonferroni posttest.

Article Snippet: HL-1 cells were seeded into six-well plates, when cells reached 70-80% confluence, they were transfected with control siRNA (sc-37007) or Mfn2 siRNA (sc-60077, both Santa Cruz Biotechnology, CA) by using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA) for 24 h. Then, HL-1 cells were treated with 200 ng/mL TM (Solarbio Life Sciences, Beijing) for the next 24 h. DS16570511 (MedChemExpress, USA) is a cell-permeable inhibitor of the mitochondrial calcium uniporter, which blocks the MCU-dependent increase of Ca 2+ influx.

Techniques: Confocal Microscopy, Fluorescence, Staining, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: Decreasing ER-mitochondria interactions by genetic downregulation of mitofusin-2 (Mfn2) protects HL-1 cells from mitochondria dysfunction. (a) Flow cytometry detected HL-1 cells that stained with DCFH-DA. (b) Quantification of ROS by DCFH-DA intensity in (a). Data represent the mean ± SEM ( n = 4 independent experiments). (c, d) JC-1 staining. The ratio of red/green fluorescence reflects changes in the mitochondrial membrane potential of HL-1 cells. Data represent the mean ± SEM ( n = 4 independent experiments). Scale bar, 100 μ m. (e, f) Analysis of oxygen consumption rate (OCR) from a Seahorse XF 24 Extracellular Flux Analyzer. Oligomycin inhibits ATP synthase, FCCP uncouples oxygen consumption from ATP production, and AA+Rotenone inhibits complexes I and III, respectively. Data represent the mean ± SEM ( n = 6 independent experiments), # p < 0.01.

Article Snippet: HL-1 cells were seeded into six-well plates, when cells reached 70-80% confluence, they were transfected with control siRNA (sc-37007) or Mfn2 siRNA (sc-60077, both Santa Cruz Biotechnology, CA) by using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA) for 24 h. Then, HL-1 cells were treated with 200 ng/mL TM (Solarbio Life Sciences, Beijing) for the next 24 h. DS16570511 (MedChemExpress, USA) is a cell-permeable inhibitor of the mitochondrial calcium uniporter, which blocks the MCU-dependent increase of Ca 2+ influx.

Techniques: Flow Cytometry, Staining, Fluorescence, Membrane

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: Mfn2 siRNA inhibited endoplasmic reticulum- (ER-) mitochondria-dependent apoptosis. (a) Cell apoptosis was evaluated by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. (b) HL-1 cells under TM stimulated for 24 hours. Mfn siRNA inhibited the Bax/Bcl-2 ratio and inhibited caspase3 and caspase9 activation (c). Data are mean ± SEM, n = 4 independent experiments, ∗ p < 0.05, # p < 0.01, ANOVA with Bonferroni posttest.

Article Snippet: HL-1 cells were seeded into six-well plates, when cells reached 70-80% confluence, they were transfected with control siRNA (sc-37007) or Mfn2 siRNA (sc-60077, both Santa Cruz Biotechnology, CA) by using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA) for 24 h. Then, HL-1 cells were treated with 200 ng/mL TM (Solarbio Life Sciences, Beijing) for the next 24 h. DS16570511 (MedChemExpress, USA) is a cell-permeable inhibitor of the mitochondrial calcium uniporter, which blocks the MCU-dependent increase of Ca 2+ influx.

Techniques: Flow Cytometry, Staining, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: Central illustration. Mfn2 deficiency in this study protected cardiomyocytes against mitochondrial depolarization/reactive oxygen species (ROS) and programmed cell death, improving atrial remodeling.

Article Snippet: HL-1 cells were seeded into six-well plates, when cells reached 70-80% confluence, they were transfected with control siRNA (sc-37007) or Mfn2 siRNA (sc-60077, both Santa Cruz Biotechnology, CA) by using a Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA) for 24 h. Then, HL-1 cells were treated with 200 ng/mL TM (Solarbio Life Sciences, Beijing) for the next 24 h. DS16570511 (MedChemExpress, USA) is a cell-permeable inhibitor of the mitochondrial calcium uniporter, which blocks the MCU-dependent increase of Ca 2+ influx.

Techniques:

Journal: Nature

Article Title: Cellular ATP demand creates metabolically distinct subpopulations of mitochondria

doi: 10.1038/s41586-024-08146-w

Figure Lengend Snippet: a . Time-lapse sequence (in minutes) illustrating the clustering of P5CS-GFP in MEFs through mitochondrial fusion. P5CS-GFP clustering was monitored 7 hours after switching cells to galactose media, capturing two distinct events (white arrows and orange arrows). See also Supplementary Video 2 . b , c . Western blot analysis of MEFs defective in mitochondrial fusion. HA-tagged MFN2 ( b ) or Flag-tagged MFN1 ( c ) cDNA was introduced to restore the ability of cells to engage in mitochondrial fusion. Actin is used as a loading control. d . Representative immunofluorescence images of endogenous P5CS and ATP5B in Mfn1 −/− MEFs expressing either empty vector (Empty) or Flag-tagged MFN1 (MFN1-Flag). MEFs were cultured in galactose medium for 8 hours. e . Western blot of DRP1 expression in wildtype, Drp1 −/− , and Drp1 −/− MEF rescued with DRP1 cDNA. Actin is used as a loading control. f . Representative immunostained images of endogenous P5CS, ATP5B, and TOM20 from Mff , Fis1 , Mid49 , Mid51 quadruple knockout (QKO) MEFs cultured in galactose medium for 8 hours. Relative pixel intensity plots corresponding to orange lines in gated area are shown on the right panels of each figure. g . Steady-state proline level measured by GC-MS in wildtype or QKO MEFs. Values are shown relative to the mean of WT MEFs. For all immunofluorescence images, DAPI was used to stain the nucleus. Scale bars are indicated as follows: 5 μm for panels a , d , and f , and 2 μm for all insets and enlarged images within panels a , d , and f . Data are presented as mean ± s.d. from n = 3 independent replicates. Statistical significance was determined using two-tailed t-tests.

Article Snippet: Mfn1 −/− (CRL-2992), Opa1 −/− (CRL-2995), Mfn1 −/− and Mfn2 −/− double knockout MEF (CRL-2994) and corresponding wildtype MEFs (CRL-2991) were obtained from the ATCC.

Techniques: Sequencing, Western Blot, Control, Immunofluorescence, Expressing, Plasmid Preparation, Cell Culture, Quadruple Knockout, Gas Chromatography-Mass Spectrometry, Staining, Two Tailed Test