Journal: Journal of Cellular and Molecular Medicine

Article Title: Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling

doi: 10.1111/j.1582-4934.2007.00111.x

Figure Lengend Snippet: ( A–D ) Merged confocal fluorescence and differential interference contrast images of sections from control ( A ), C2C12/GFP-transplanted ( B ) and C2C12/RLX-transplanted ( C, D ) hearts. Sections were stained with anti-GFP antibodies (red) to identify the engrafted myoblasts and counter-stained with Syto3 (green) to reveal nuclei ( A–C ).In ( D ), the sections were stained with antibodies against the skeletal muscle-specific isoform of a-sarcomeric actin. Myoblast-injected hearts show immunoreactive cells in the post-infarcted zone ( B–D ), mainly located around blood vessels (asterisks) and scattered within the extracel-lular matrix. No labelled cells can be detected in the controls. C2C12/RLX-implanted hearts contain significantly more GFP-immunoreactive cells than the C2C12/GFP-implanted ones. ( E–G ) Confocal micrographs of sections from C2C12/RLX-treated hearts, immunostained for V-CAM ( E ) and ICAM ( F, G ). Both molecules (green) are expressed by endothelial cells of microvessels in the post-infarcted zones ( E, F ); by contrast, no ICAM staining is observed in the surrounding viable myocardium. Nuclei are counterstained with propidium iodide (red). Bars = 20 μm.

Article Snippet: Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM containing 10% foetal bovine serum (Sigma, Milan, Italy) Cells were transduced with a lentiviral vector bicistronically expressing human preprore-laxin 2 cDNA [ ] and eGFP gene or just eGFP, under a cytomegalovirus (CMV) promoter and termed C2C12/RLX and C2C12/GFP, respectively.

Techniques: Fluorescence, Control, Staining, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling

doi: 10.1111/j.1582-4934.2007.00111.x

Figure Lengend Snippet: Immunoelectron micrographs of C2C12 myoblasts grafted in the post-infarcted heart, immunostained with anti-RLX antibodies (revealed by 5 nm gold particles). In the upper panel, a C2C12/RLX myoblast shows clear-cut cytoplasmic RLX immunoreactivity (details in the insets).In the lower panel, a C2C127GFP myoblast shows no immunolabelling (detail of the RER area in the inset). Both cells show a discontinuous basement membrane (arrows) and are surrounded by a loose extracellular matrix. Bar = 0.5 μm.

Article Snippet: Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM containing 10% foetal bovine serum (Sigma, Milan, Italy) Cells were transduced with a lentiviral vector bicistronically expressing human preprore-laxin 2 cDNA [ ] and eGFP gene or just eGFP, under a cytomegalovirus (CMV) promoter and termed C2C12/RLX and C2C12/GFP, respectively.

Techniques: Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling

doi: 10.1111/j.1582-4934.2007.00111.x

Figure Lengend Snippet: Upper panels. Light micro-graphs of sections from control ( A ), C2C12/GFP- ( B ) and C2C12/RLX-transplanted hearts ( C ), stained with Van Gieson for collagen. A marked reduction of the extent of fibrosis can be seen in the ischaemic scars of the myoblast-transplanted hearts; of note, fibrosis is particularly attenuated in the hearts receiving C2C12/RLX cells. Bar = 20 μm. Centre panel. Sclerosis index (optical x volume density of collagen fibres) in the post-infarcted myocardi-um, estimated by computer-aided mor-phometry; ( a ) P <0.05 versus controls; ( b ) P <0.001 versus controls; ( c ) P <0.001 versus C2C12/GFP. Lower panels. Electron micrographs of fibrob-lasts from control ( D ) C2C12/GFP- ( E ) and C2C12/RLX-transplanted hearts ( F ). In the control, collagen microfibrils form large bundles adjacent to the cell's plasma membrane (inset), as occurs during de novo fibre assembly; in the C2C12/GFP-transplanted hearts, and even more in the C2C12/RLX-transplanted ones, collagen microfibrils are scattered and loosely arranged. Bar = 0.5 μm.

Article Snippet: Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM containing 10% foetal bovine serum (Sigma, Milan, Italy) Cells were transduced with a lentiviral vector bicistronically expressing human preprore-laxin 2 cDNA [ ] and eGFP gene or just eGFP, under a cytomegalovirus (CMV) promoter and termed C2C12/RLX and C2C12/GFP, respectively.

Techniques: Control, Staining, Clinical Proteomics, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling

doi: 10.1111/j.1582-4934.2007.00111.x

Figure Lengend Snippet: Upper panel. Matrix metallopro-tease (MMP) activity evaluated by gelatin zymography on cardiac tissue samples from control (n = 2, optical density: 36±1), C2C12/GFP-transplanted (n = 2, optical density: 92±4) and C2C12/RLX-transplanted swine (n = 4, optical density: 106±13). Centre and lower panel. MMP activity on conditioned media from: primary cultures of swine cardiac cells co-cultured with C2C12/GFP (1) or C2C12/RLX myoblasts (2), primary cultures of swine cardiac cells incubated with medium (3) or with H2 RLX (4), C2C12/GFP (5) and C2C12/RLX myoblasts (6). Densitometric analysis of MMP activity, expressed as arbitrary optical density units normalized to protein content. Molecular weights of the collagen lysis bands are consistent with MMP-9 and MMP-2. Transfection or treatment with RLX preferentially up-regulated MMP 2 activity. ( a ), P <0.05 versus the co-cultures of cardiac cells and C2C12/GFP; ( b ) P <0.05 versus the counterparts without RLX; ( c ) P <0.05 versus cardiac cells treated with exogenous RLX.

Article Snippet: Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM containing 10% foetal bovine serum (Sigma, Milan, Italy) Cells were transduced with a lentiviral vector bicistronically expressing human preprore-laxin 2 cDNA [ ] and eGFP gene or just eGFP, under a cytomegalovirus (CMV) promoter and termed C2C12/RLX and C2C12/GFP, respectively.

Techniques: Activity Assay, Zymography, Control, Cell Culture, Incubation, Lysis, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling

doi: 10.1111/j.1582-4934.2007.00111.x

Figure Lengend Snippet: Upper and centre panels. Light micrographs of the microvascular network in control ( A ), C2C12/GFP- ( B ) and C2C12/RLX-transplanted hearts ( C ), and morphometric assessment of microvascular density. Both C2C12/ GFP- and C2C12/RLX-injected hearts exhibit higher microvascular density than the control hearts. Of note, C2C12/RLX-injected hearts show significantly higher microvascular density than C2C12/GFP-injected ones. ( a ) P <0.05 versus control;( b ) P <0.01 versus control;( c ) P <0.05 versus C2C12/ GFP. Lower panel. RT-PCR assay for porcine VEGF transcripts in scar tissue samples from controls (lanes 1, 2), C2C12/GFP- (lanes 3,4) and C2C12/ RLX-transplanted hearts (lanes 5, 6). Images are representative of five different animals per experimental groups. Porcine VEGF mRNA is absent in the control samples, is slightly expressed in one of the two samples of C2C12/GFP-injected hearts, and clearly detectable in both the samples from C2C12/RLX-injected hearts.

Article Snippet: Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM containing 10% foetal bovine serum (Sigma, Milan, Italy) Cells were transduced with a lentiviral vector bicistronically expressing human preprore-laxin 2 cDNA [ ] and eGFP gene or just eGFP, under a cytomegalovirus (CMV) promoter and termed C2C12/RLX and C2C12/GFP, respectively.

Techniques: Control, Injection, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling

doi: 10.1111/j.1582-4934.2007.00111.x

Figure Lengend Snippet: Myocardial performance index (MPI), ejection fraction (EF), left ventricular end-diastolic and end-systolic diameters (LVEDD, LVESD) evaluated in basal conditions (black bars), 1 month after the induction of myocardial infarction (post-AMI; striped bars) and 1 month after cell transplantation (post-cellular cardiomyoplasty [CCM]; open bars).Myoblast implantation improves cardiac function. C2C12/RLX myoblasts are more effective than C2C12/GFP ones. Significance of differences (one-way ANOVA): MPI: ( a ) P <0.001 versus basal; ( b ) P <0.001 versus post-AMI; ( c ) P <0.01 versus controls; ( d ) P <0.001 versus controls; ( e ) P <0.05 versus C2C12/GFP. EF: ( a ) P <0.01 versus basal; ( b ) P <0.001 versus post-AMI; ( c ) P <0.001 versus controls. LVEDD: ( a ) P <0.05 versus basal; ( b ) P <0.01 versus basal; ( c ) P <0.01 versus controls. LVESD: ( a ) P <0.01 versus basal & post-AMI; ( b ) P <0.05 versus controls;( c ) P <0.001 versus controls.

Article Snippet: Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM containing 10% foetal bovine serum (Sigma, Milan, Italy) Cells were transduced with a lentiviral vector bicistronically expressing human preprore-laxin 2 cDNA [ ] and eGFP gene or just eGFP, under a cytomegalovirus (CMV) promoter and termed C2C12/RLX and C2C12/GFP, respectively.

Techniques: Transplantation Assay

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Schematic overview of experimental strategy for monitoring the 3D nuclear position of the MyoD1 locus in living myoblasts by inserting a 64x LacO array downstream of MyoD1 coupled with expression of GFP-LacR. b Representative 3D reconstructed z stack of images showing intranuclear 3D position of the MyoD1 locus in proliferating myoblasts (green dots). Scale bars are indicated on images. See also Supplementary Movie . c Immunofluorescence microscopy of proliferating and 4-days differentiated MyoD1 and Pax7 reporter myoblasts. MyoD1 and Pax7 loci are indicated by yellow arrowheads. Lamin A/C staining (red) marks nuclear border. Scale bars, 10 μm. d Schematic drawing depicting the analysis of the radial distance of gene loci to the nuclear periphery and normalization based on nuclear height. e Density plots of the normalized radial distance of loci to the periphery. Left: Distribution of MyoD1 in proliferating (red) and differentiating (day 4, green) C2C12 myoblasts. n prolif = 106, n diff = 120, p = 1 × 10 −4 . Right: Distribution of MyoD1 and Pax7 loci in proliferating myoblasts. n MyoD1 = 208, n Pax7 = 79, p = 6.3 × 10 −4 . p values for loci were calculated using two-sample, two-sided KS test and corrected for multiple testing using Hochberg method. Numeric values displayed on violin plots represent median values. f Normalized radial position distribution of data shown in ( e ) displayed in violin plots. H3K9me2-marked region (gray-shaded horizontal bar) represents peripheral heterochromatin layer as measured in ( g ). **** p = 1 × 10 − 4 , *** p = 6.3 × 10 −4 p values were calculated using two-sample, two-tailed KS test and corrected for multiple testing using Hochberg method. g H3K9me2 immunofluorescence staining of MyoD1 C2C12 reporter myoblasts. Width of H3K9me2-marked layer is measured in rendered images (middle panel, yellow dots and lines illustrate measurement method for heterochromatin layer), bar graph depicts normalized mean thickness ± SEM of heterochromatin layer measured at 100 different locations. Scale bars, 10 μm. Images in ( c ) were processed with Fiji and in ( b ) and ( g ) with Imaris. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Immunoblot analysis of total cell extracts of proliferating wildtype and knockout C2C12 clones using antibodies to the indicated antigens. Normalized radial distance of MyoD1 to the nuclear border in wildtype C2C12 myoblasts versus cells with ( b ) single knockouts, and ( c ) double or triple knockouts of indicated proteins. H3K9me2-marked heterochromatin layer is shown as gray-shaded bar. Dotted line indicates that experiments were performed separately using slightly different laser settings. n Wt = 208, n emerin KO = 333, n LAP2β KO = 140; n Wt = 141, n LA/C KO = 403, n LBR KO = 283; n Wt = 722, n LA/C&LBR KO = 453, n LA/C, LBR&Emd KO = 300. * p Emd KO = 0.02, remaining p values are non-significant ( p > 0.05; two-sample, two-tailed KS test, Hochberg correction for multiple testing). Numeric values displayed on violin plots represent median values. LA/C lamin A/C, Emd emerin. d Localization of emerin assessed by immunofluorescence microscopy in control and lamin A/C-LBR double knockout myoblasts. Scale bars, 10 μm. Images processed with Fiji. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Western Blot, Knock-Out, Clone Assay, Two Tailed Test, Immunofluorescence, Microscopy, Control, Double Knockout

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Immunostaining and 3D reconstruction of H3K9me3-marked heterochromatin in wildtype and lamin A/C-LBR double knockout C2C12 cells. Scale bars, 10 μm. b Graphs show quantification of mean area per intranuclear H3K9me3-positive spot (left), and number of intranuclear foci per cell (right). Bar graphs represent mean ± SD. n Wt = 40, n KO = 31, n KO2 = 31 (single data points are displayed). Left graph: H(2) = 11.72, ** p KO = 0.0096, ** p KO2 = 0.0058; right graph: H(2) = 52.07, **** p KO = 9.8 × 10 -8 , **** p KO2 = 6.6 × 10 −11 (Kruskal-Wallis test). c Nucleoplasmic over peripheral H3K9me3 signal ratio determined using line intensity profiles across the nucleus in wildtype and lamin A/C-LBR double knockout cells. Line graph on the right shows a representative H3K9me3 fluorescence intensity plot measured along the dashed lines in the images shown in ( a ). Bar graph displays mean values ± SD ( n Wt = 4, n KO = 5). t (7) = 8.31, **** p = 7.1 × 10 −5 (two-tailed t-test). d Lamin B1 immunostaining of indicated cell lines. Scale bars, 10 μm. e Lamin B1 ChIP-qPCR analyses demonstrating loss of lamin B1 binding to LADs at the nuclear periphery of lamin A/C-LBR double knockout cells. Data represent mean values ± SEM of three biological replicates. Horizontal dashed line indicates the signal obtained using unspecific IgG antibodies in control ChIP. The genomic positions of tested LADs are indicated in the table. f Representative confocal images of fluorescence in-situ hybridization (FISH) signal using a GFP-labeled BAC probe for a LAD region (green dots). Dashed line indicates nuclear border. Scale bars, 10 μm. g Radial distance of LAD region to the nuclear periphery upon depletion of lamin A/C and LBR as detected by FISH. n Wt = 909, n KO = 666, **** p = 7.6 × 10 -4 (two-sample, two-tailed KS test). Images in ( a ) processed with Imaris, and in ( d ) and ( f ) with Fiji. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Immunostaining, Double Knockout, Fluorescence, Two Tailed Test, ChIP-qPCR, Binding Assay, Control, In Situ Hybridization, Labeling

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Schematic overview of the strategy for generating multiple protein knockout cell lines. MyoD1 reporter C2C12 myoblasts constitutively expressing Cas9 were transfected with three synthetic sgRNAs targeting the gene of interest. Depletion of various candidate proteins was performed consecutively. b Detection of normalized radial distance of MyoD1 to periphery in proliferating myoblasts following depletion of indicated protein(s). Violin plots were generated from at least 450 data points. n Wt = 722, n 4KO = 454, n 3KO = 898, n 2KO = 685, n NET39 KO = 500; n Tmem38a KO = 571, n WFS1 KO = 517. *** p 4KO = 2 × 10 −4 , **** p 3KO = 3.2 × 10 −6 , **** p 2KO = 7.9 × 10 −5 , p NET39 KO = 0.6; *** p Tmem38a KO = 2 × 10 −4 , **** p WFS1 KO = 1 × 10 −6 (two-sample two-tailed KS test, Hochberg correction for multiple testing). Numeric values displayed on the violin plot represent median values. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Knock-Out, Expressing, Transfection, Generated, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Ablation of Aster-B impairs uptake of cholesterol and cholesterol esters. (A) Time-lapse confocal imaging analysis depicting the cholesterol uptake in C2C12 vector control (VC) and Aster-B knockout (KO) cells. Cells were depleted of endogenous cholesterol using 0.5% methyl-β-cyclodextrin treatment for 2 hr and then treated with 50 μM of 25-NBD-Cholesterol for the times indicated. Nuclei are stained with Hoechst 33342. (B) Time-lapse confocal imaging analysis depicting the cholesterol ester uptake in C2C12 VC and Aster-B KO cells. Cells were starved in KRPH buffer for 30 min and treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for the times indicated. Nuclei are stained with Hoechst 33342. (C) Fluorescence density analysis of green fluorescent cholesterol treated VC and KO cells at 30 minutes, n=10. (D) Fluorescence density analysis of CholEsteryl BODIPY™ 542/563 C11-treated VC and KO cells at 12 min n = 10. ∗∗∗∗p < 0.0001 by student's t-test.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Imaging, Plasmid Preparation, Knock-Out, Staining, Fluorescence

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Aster-B deficiency impairs the transport of cholesterol and fatty acids derived from cholesterol esters into mitochondria. (A) Confocal imaging analysis depicting the mitochondrial transport of cholesterol ester in vector control (VC) and Aster-B knockout (KO) cells. Cells were starved in KRPH buffer for 30 min and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. Digitonin-treated cells were starved in KRPH buffer for 20 min and then pre-treated with 10 μM of digitonin for 10 min, washed twice in KRPH buffer, and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. Mitochondria and nuclei were stained by Mitotracker-Green and Hoechst 33342, respectively. (B) Pearson's Co-localization Coefficient of Mitotracker Green and CholEsteryl BODIPY™ 542/563 C11 in the VC and KO cells. n = 20. ∗∗∗∗p < 0.0001 by student's t-test. (C) Pearson's Co-localization Coefficient of Mitotracker Green and CholEsteryl BODIPY™ 542/563 C11 in vehicle and digitonin-treated Aster-B KO cells. n = 20. (D) Confocal images of C2C12 cells pre-treated with vehicle or 300 nM of etomoxir for 6 h. Cells were then starved for 30 min and treated with CholEsteryl BODIPY™ 542/563 C11 for 10 min. Mitochondria were stained with Mitotracker-Green. (E) Pearson's Co-localization Coefficient of Mitotracker-Green and CholEsteryl BODIPY™ 542/563 C11 in D. n = 20. ∗∗∗∗p < 0.0001 by student's t-test. (F) Confocal imaging analysis depicting the co-localization of cholesterol with mitochondria in VC and Aster-B KO cells. Cells were treated with MCD, permeabilized by digitonin, and then incubated with 16:0 TopFluor® cholesterol. Mitochondria were stained with Mitotracker-Red. (G) Pearson's Co-localization Coefficient of Mitotracker-Red and 16:0 TopFluor® cholesterol in F. n = 20. ∗∗∗p < 0.001 by student's t-test.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Derivative Assay, Imaging, Plasmid Preparation, Knock-Out, Staining, Incubation

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: MTS is required for the tethering of Aster-B at ER and mitochondrial contact sites. (A) Schematic representation of Aster-B protein showing deleted region (2–31) of plasmid with MTS highlighted in yellow. (B–C) Confocal images of C2C12 Aster-B knockout (KO)cells transfected with GFP labeled Aster-B or Δ2-31-Aster-B and an ER localized DsRed-ER protein (B) or a mitochondria resident protein, Mito-DsRed (C), respectively. Cells were starved for 30 min in KRPH buffer followed by 10 min of 10 μM of CholEsteryl BODIPY™ 542/563 C11 stimulation. (D–E) Subcellular fractionation and Western blot analysis of 293A cells transfected with either wild-type (WT) or Δ2-31 Aster-B plasmids. Cells were starved for 30 min in KRPH buffer and then treated with 10 μM of non-fluorescent cholesterol esters for 10 min. Micro, microsome; Cr. Mito, crude mitochondria, pure mito, pure mitochondria; cyto, cytosol. (F) Densitometric analysis showing the fold difference of WT Aster-B to Δ2-31 in the MAM, using Bip as an internal control, n = 3. ∗∗∗∗p < 0.0001 by two-way ANOVA. (G) Densitometric analysis showing the fold difference of WT Aster-B to Δ2-31 in the purified mitochondrial fraction using Tom20 as an internal control n = 3. ∗∗∗p < 0.001 by two-way ANOVA.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Plasmid Preparation, Knock-Out, Transfection, Labeling, Fractionation, Western Blot, Purification

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Deletion of MTS of Aster-B impairs mitochondrial cholesterol trafficking. (A–B) Confocal imaging analysis depicting the mitochondrial cholesterol transport in C2C12 Aster-B knockout (KO) cells re-expressing GFP-Aster-B WT (A) or Δ2-31 mutant (B). Mitochondria were labeled by transfecting cells with mito-BFP. Cells were starved for 20 min in KRPH buffer, followed by 10 min of permeabilization with 10 μM of digitonin, and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. (C–E) Total cholesterol level (C) and cholesterol levels in pure mitochondria (D) and MAM (E) in Aster-B KO cells re-expressing GFP-Aster-B WT or Δ2-31 mutant. Cells were treated with MCD (-CHOL) or permeabilized by digitonin after MCD treatment and re-incubated with 50 μM of cholesterol (+CHOL). n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001 by two -ANOVA.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Imaging, Knock-Out, Expressing, Mutagenesis, Labeling, Incubation

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Arf1 is required for cholesterol transport into mitochondria. (A) Confocal imaging analysis depicting the mitochondrial cholesterol transport in HeLa vector control (VC) and Arf1 knockout (Arf1-KO) cells. Cells were starved in KRPH buffer for 30 min and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. (B) Confocal imaging analysis depicting the mitochondrial cholesterol transport in C2C12 cells treated with 10 μM of Exo-2 for 2 h followed by incubation with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. Nuclei were stained with Hoechst 33342, and mitochondria were stained with Mitotracker-Green. (C–E) Total cholesterol level (C) and cholesterol levels in pure mitochondria (D) and MAM (E) in HeLa VC and Arf1 KO cells. Cells were treated with MCD (-CHOL), or pemeabilized by digitonin after MCD treatment and re-incubated with 50 μM of cholesterol (+CHOL). n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Imaging, Plasmid Preparation, Knock-Out, Incubation, Staining

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Ablation of Aster-B or inhibition of Arf1 causes mitochondrial dysfunction. (A) Seahorse XF analysis showing oxygen consumption rate (OCR) of C2C12 vector control with vehicle (VC) or 10 μM of Exo-2 (VC Exo-2) and Aster-B knockout (KO) cells treated with MCD for 2 hr and then treated with 25 μM non-fluorescent cholesterol ester for 1 h. Cells were treated with vehicle or 10 μM of Exo-2 throughout the assay. (B) Basal, (C) maximum, (D) ATP-linked respiration of the Seahorse XF analysis is shown. n = 5. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Inhibition, Plasmid Preparation, Knock-Out

Journal: JCI Insight

Article Title: Role of a TRIM72 ADP-ribosylation cycle in myocardial injury and membrane repair

doi: 10.1172/jci.insight.97898

Figure Lengend Snippet: (A) Western blot analysis of C2C12 cell lysates after TRIM72 (shTRIM72), ARH1 (shARH1), ART1 (shART1), and ARH1 plus ART1 (shARH1/ART1) shRNA transformation, demonstrating decreased protein expression compared with control scrambled shRNA (shCont.) transfectant. (B and C) Laser wounding assays were performed with C2C12 cells transformed with TRIM72-GFP in control scrambled shRNA (n = 35), ARH1 shRNA (n = 34), ART1 shRNA (n = 31), TRIM72 shRNA (n = 35), or double knockdown for shART1 and shARH1 (n = 31) with or without overexpression with WT TRIM72-GFP or mutant TRIM72(R207K,R260K)-GFP (n = 37) (B) (Supplemental Videos 5–10). (C) Measurements of GFP intensity at injury sites. (D and E) Scratch wound healing assays were performed with cells transformed with shCont., shARH1, shART1, shTRIM72, or shART1/ARH1 knockdown cells with or without overexpression with mutant TRIM72(R207K,R260K)-GFP or WT TRIM72–GFP. Scale bar: 1 mm. (E) Quantification of the area covered by cells at each time is shown as percentage of time 0 wound area (n = 12). (F) Western blots are shown for detection of ADP-ribosylated TRIM72 coimmunoprecipitated with active or inactive Af1521 macrodomain–GST from C2C12 myotube lysates with scratching with needles. The amount of ADP-ribosylated TRIM72 was increased after scratching of C2C12 cells transformed with control shRNA or ARH1 shRNA. In addition, in scratched C2C12 myocytes, Af1521 macrodomain–GST pull-down revealed that WT TRIM72-GFP, but not mutant-TRIM72(R207K,R260K)-GFP, was ADP-ribosylated. ADPr-TRIM72 coimmunoprecipitated with Af1521 macrodomain–GST was detected by Western blotting (WB) with anti-TRIM72 antibody. Data shown are representative of 3 experiments. Data are mean ± SEM. Open symbols are values significantly different (P < 0.05) from those of shCont. cells by 2-way ANOVA, followed by Bonferroni’s post hoc tests.

Article Snippet: C2C12 cells were prepared that stably expressed ARH1, ART1, TRIM72, and control shRNA vectors (Origene) and transiently overexpressed pEGFP-N1-TRIM72 and pEGFP-N1-TRIM72 (R207K, R260K) vectors.

Techniques: Western Blot, shRNA, Transformation Assay, Expressing, Transfection, Over Expression, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: Protein O -Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

doi: 10.1128/MCB.00890-14

Figure Lengend Snippet: Characterization of the Pofut1 knockdown C2C12 cell line. (A) Pofut1 expression in MB of Pofut1 shRNA C2C12 clones. Histograms showed Pofut1 expression relative to Gapdh for each clone compared to that in WT C2C12 cells. (B) Relative quantities of Pofut1 expression in WT C2C12 and Po− cells. In both cell lines, fold changes are expressed relative to the value at 0 h of WT C2C12 cells. (C) Western blot analyses of Pofut1 expression in WT C2C12, control shRNA, and Po− cells. Histograms represent quantification of Pofut1 band intensity relative to Gapdh. All ratios were calibrated relative to time zero of WT C2C12 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: A control C2C12 cell line (Ctrl shRNA) was obtained by electroporation of a pSilencer 2.1-U6-neomycin vector containing a negative-control siRNA template (Ambion, Life Technologies) into C2C12 cells.

Techniques: Expressing, shRNA, Clone Assay, Western Blot