Journal: Scientific Reports

Article Title: Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF 164 in skeletal muscle

doi: 10.1038/srep21546

Figure Lengend Snippet: In vivo expression of the Vegfa and Pdgfb genes, discriminating between the delivered and endogenous transcripts, was quantified in calf muscles 4 and 7 days after implantation with control cells (Ctrl) and myoblast clones expressing medium or high levels of VEGF alone or with PDGF-BB (V and VIP, respectively). Data are shown as mean ± SEM (n = 4 muscles/condition); *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (ANOVA with Sidak multiple comparisons test after data normalization by logarithmic transformation).

Article Snippet: To determine the expression of the other genes the following TaqMan gene expression assays were used from Applied Biosystems (Applied Biosystems, Zug, Switzerland): murine Pdgfb (Mm00440678_s1), human Pdgfb (Hs00234042_m1) and murine Gapdh housekeeping gene (Mm03302249_g1).

Techniques: In Vivo, Expressing, Muscles, Control, Clone Assay, Transformation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension

doi: 10.1152/ajplung.00531.2016

Figure Lengend Snippet: CH leads to an increase in lung T helper 17 (TH17) cells without T regulatory cells being affected. Lungs were removed from vehicle- and SR1001 (25 mg·kg−1·day sc−1)-treated mice after 5 days of normoxic or CH (CH 5d) exposure. Single-cell suspensions from the lungs were labeled with anti-CD3, anti-CD4, and anti-IL-17A or FoxP3. A: representative scatter plots. Cells were gated based on forward and side scatter and CD3 CD4. B: summary of the %CD4+ IL-17A+ or CD4+ FoxP3+ cells relative to total CD3+ cells normalized to the average of the vehicle-treated normoxic group of each set of experiments. C: representative scatter plots of CD3+, CD4+ T helper cells from lungs from mice exposed to normoxia or 5 days of CH. D: summary of the %CD3+ T cells relative to total cells and CD3+, CD4+, and T helper cells relative to total CD3+ cells. Values are means ± SE; n = no. of animals, *P < 0.05 vs. normoxia vehicle; #P < 0.05 vs. CH vehicle, analyzed by 2-way ANOVA, followed by multiple-comparison Student-Newman-Keuls test.

Article Snippet: Images were captured every hour for a total of 60 h. Cells were incubated with or without 100 ng/ml of Eschericia coli -derived recombinant mouse IL-17A (R & D Systems).

Techniques: Labeling

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension

doi: 10.1152/ajplung.00531.2016

Figure Lengend Snippet: The adoptive transfer of TH17 cells causes spontaneous PH in RAG1−/− mice. CD4+ T cells were purified from IL-17A enhanced green fluorescent protein (EGFP) mice and cultured under TH17-polarizing conditions. TH17 cells were purified by fluorescence-activated cell sorting and injected into RAG1−/− mice. After 14 days, mice remained in normoxia or were exposed to CH for 21 days and compared with mice that did not receive TH17 cells. A: RVSP. B: %wall thickness. C: Fulton’s index. D: hematocrit. E: representative images of lung sections immunostained for CD3 from mice receiving adoptive transfer or a RAG1−/− mouse that received no adoptive transfer. Scale bars, 100 µm. F: flow cytometric analysis of TH17 cells from the spleens of mice that received an adoptive transfer. Values are means ± SE; n = no. of animals. *P < 0.05 vs. normoxia; #P < 0.05 vs. normoxia No AT; &P < 0.05 vs. CH No AT. Analyzed by 2-way ANOVA, followed by multiple comparisons Student-Newman-Keuls test.

Article Snippet: Images were captured every hour for a total of 60 h. Cells were incubated with or without 100 ng/ml of Eschericia coli -derived recombinant mouse IL-17A (R & D Systems).

Techniques: Adoptive Transfer Assay, Purification, Cell Culture, Fluorescence, FACS, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension

doi: 10.1152/ajplung.00531.2016

Figure Lengend Snippet: IL-17A stimulates migration but does not affect mouse pulmonary arterial smooth muscle cell number in culture. A: %wound density in the presence or absence of IL-17A (100 ng/ml). B: confluency density normalized to time 0 in the presence or absence of IL-17A (100 ng/ml) in serum-starved cells. Growth medium was used a positive control. *P < 0.05, cells from n = 4 mice, 2-way repeated-measures ANOVA, followed by multiple-comparison Student-Newman-Keuls test.

Article Snippet: Images were captured every hour for a total of 60 h. Cells were incubated with or without 100 ng/ml of Eschericia coli -derived recombinant mouse IL-17A (R & D Systems).

Techniques: Migration, Positive Control

Journal: PLoS ONE

Article Title: Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study

doi: 10.1371/journal.pone.0104888

Figure Lengend Snippet: RL-14 human immortalized fetal cardiomyocytes and rat neonatal primary cardiomyocytes (Rat CM) were cultured with same conditions as we did for H9c2 cells, and (A) cell viabilities, (B) LDH leakages, (C) ROS generations and (D) [Ca2+] i were evaluated at 72 hour, 24 hour, 6 hour and 90 minute, respectively. Values represent mean ± S.D. (n = 4) from quadruplicate samples for each treatment at varying treatment conditions. * p<0.05.

Article Snippet: H9c2 rat fetal cardiomyoblasts (ATCC CRL-1446), RL14 human fetal cardiomyocytes (ATCC PTA-1499) and rat neonatal primary cardiomyocytes (Lonza, Allendale, NJ, USA) were grown in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) fetal bovine serum in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study

doi: 10.1371/journal.pone.0104888

Figure Lengend Snippet: (A) Rat neonatal primary cardiomyocytes (CM) and H9c2 cardiomyoblasts were cultured with reduced FBS (1%) for 72 hours, with the indicated conditions. Cell lysates from the culture were subject to Western blotting using antibodies against phosphorylated or total platelet-derived growth factor receptor β-subunit (PDGFRβ). An image of the gel stained with GelCode Blue dye (Thermo) after transfer was shown as a loading monitor. The histograms show the densitometric scanning results. (B) Quiescent H9c2 cells were incubated with 0.1 or 1.0 mM metformin (Met) for 5 minutes with or without forskolin (1 µM). After the treatment, PI3K activities were determined as described in . The histograms show the densitmetric scanning results. PIP, phosphoinositide 3-phosphate. Ori, origin of migration in thin-layer chromatography. (C) H9c2 cells were cultured for 72 hours with reduced FBS (1%), under the indicated conditions with or without AG1296 (10 µM). The cell viabilities were evaluated as described. * p<0.05, # p<0.05 vs CTR, $ p<0.05 vs Dox 10 nM + Met 0.1 mM. Values represent mean ± S.D. (n = 4) from quadruplicate samples for each treatment at varying treatment conditions.

Article Snippet: H9c2 rat fetal cardiomyoblasts (ATCC CRL-1446), RL14 human fetal cardiomyocytes (ATCC PTA-1499) and rat neonatal primary cardiomyocytes (Lonza, Allendale, NJ, USA) were grown in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) fetal bovine serum in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Cell Culture, Western Blot, Derivative Assay, Staining, Incubation, Migration, Thin Layer Chromatography

Journal: PLoS ONE

Article Title: Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study

doi: 10.1371/journal.pone.0104888

Figure Lengend Snippet: Low dose (0.1 mM) Met induces moderate AMPK activation which is followed by sequential activations of PKA, Src and PDGFR and results in protection of the cells from Dox toxicity. High dose (1.0 mM) Met also leads to the sequential activations of AMPK, PKA and Src. With both dosages of the Met treatment, Dox induced ROS generations and [Ca 2+ ] i levels are suppressed. However, the excessive activities of AMPK with the high dose Met causes a suppression of PDGFR expression in cardiomyocytes and the protective effect of low dose Met is abrogated.

Article Snippet: H9c2 rat fetal cardiomyoblasts (ATCC CRL-1446), RL14 human fetal cardiomyocytes (ATCC PTA-1499) and rat neonatal primary cardiomyocytes (Lonza, Allendale, NJ, USA) were grown in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) fetal bovine serum in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Activation Assay, Expressing

Journal: JCI Insight

Article Title: EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

doi: 10.1172/jci.insight.157701

Figure Lengend Snippet: ( A ) Representative laser Doppler perfusion images in EPCR fl/fl Tie2.Cre mice ( n = 11) and EPCR fl/fl littermate controls ( n = 10) immediately before and after, as well as on, day 7, day 14, day 21, and day 28 after induction of hindlimb ischemia. For technical reasons, 1 animal in each group could not be imaged at day 21. ( B ) Quantitative analysis of the laser signal (expressed as % of the contralateral, nonischemic site). * P < 0.05, *** P < 0.001, and **** P < 0.0001 versus values immediately after surgery; ## P < 0.01 versus EPCR fl/fl controls at the same time point; 2-way ANOVA, Sidak’s multiple-comparison test. ( C ) Representative immunofluorescence microscopy images after visualization of CD31 expression (red) in nonischemic and ischemic hindlimbs of EPCR fl/fl Tie2.Cre mice and control littermates. DAPI + cell nuclei appear blue. Scale bar: 10 μm. ( D ) Quantitative analysis of CD31-immunopositive cells per muscle fiber; n = 7 biological replicates. * P < 0.05 and *** P < 0.001 versus nonischemic control muscle; # P < 0.05 versus EPCR fl/fl littermates; 2-way ANOVA, Sidak’s multiple-comparison test. ( E ) Representative bright-field images of CD31 + endothelial cells isolated from EPCR fl/fl and EPCR fl/fl Tie2.Cre littermate mice and subjected to the spheroid assay; n = 4 biological replicates per 2 experimental repeats. Scale bar: 10 μm. ( F and G ) Quantitative analysis of the number of sprouts ( F ) and total length of the sprouts ( G ) migrated out of the spheroids. ** P < 0.01 and **** P < 0.0001 versus EPCR fl/fl littermate control mice. Data were analyzed using Student’s t test.

Article Snippet: Capillary density in the gastrocnemius muscle was assessed 28 days after induction of unilateral hindlimb ischemia on 5 μm–thick, acetone-fixed frozen sections (for 10 minutes at –20°C) stained with lycopersicon esculentum lectin or monoclonal rat antibodies against murine CD31 (Pecam1; Santa Cruz Biotechnology; clone MEC13.3; sc-18916; dilution, 1:50) to label immature, angiogenic endothelial cells , as well as antibodies against CD41 (Itga2b, Biozol; 11-763-C100; dilution, 1:100) to label platelets, eNOS (D9A5L; Cell Signaling Technology; 32027; dilution, 1:100), EPCR (R&D Systems; AF2245; dilution, 1:100), fibrinogen (Abcam; clone EPR18145-84; ab189490; dilution, 1:100), HBA (LifeSpan BioSciences; LS-B10549 1:100), PDGFRB (Abcam; ab32570; dilution, 1:100), and α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich; A2547; dilution, 1:50) to label smooth muscle cells or TF (R1073; dilution, 1:100) ( ).

Techniques: Comparison, Immunofluorescence, Microscopy, Expressing, Control, Isolation

Journal: JCI Insight

Article Title: EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

doi: 10.1172/jci.insight.157701

Figure Lengend Snippet: ( A and B ) Representative high resolution immunofluorescence images ( A ) and quantitative analysis ( B ) after staining of ischemic and nonischemic hindlimb muscles of EPCR fl/fl Tie2.Cre mice and EPCR fl/fl littermate controls with antibodies against TF (red) and CD31 (green). Two-way ANOVA, Sidak’s multiple-comparison test. * P < 0.05 versus the nonischemic, contralateral hindlimb; # P < 0.05 and ## P < 0.01 versus EPCR fl/fl control littermate mice; n = 6 biological replicates. ( C and E ) Representative laser Doppler perfusion images in EPCR fl/fl Tie2.Cre ( n = 3 control, n = 2 NAPc2 treatment) ( C ) and littermate EPCR fl/fl control mice ( n = 3) ( E ) immediately before and after, as well as on, day 21 after induction of hindlimb ischemia, treated with PBS as a control or NAPc2 (0.5 mg/kg body weight; directly after surgery and afterward every 2 days). ( D and F ) Quantitative analysis of the laser signal (expressed as % of the contralateral, nonischemic site) for EPCR fl/fl Tie2.Cre ( D ) and EPCR fl/fl littermate controls ( F ). ** P < 0.01 and *** P < 0.001 versus values immediately after surgery; # P < 0.05 versus untreated controls at the same time point; 2-way ANOVA, Sidak’s multiple-comparison test.

Article Snippet: Capillary density in the gastrocnemius muscle was assessed 28 days after induction of unilateral hindlimb ischemia on 5 μm–thick, acetone-fixed frozen sections (for 10 minutes at –20°C) stained with lycopersicon esculentum lectin or monoclonal rat antibodies against murine CD31 (Pecam1; Santa Cruz Biotechnology; clone MEC13.3; sc-18916; dilution, 1:50) to label immature, angiogenic endothelial cells , as well as antibodies against CD41 (Itga2b, Biozol; 11-763-C100; dilution, 1:100) to label platelets, eNOS (D9A5L; Cell Signaling Technology; 32027; dilution, 1:100), EPCR (R&D Systems; AF2245; dilution, 1:100), fibrinogen (Abcam; clone EPR18145-84; ab189490; dilution, 1:100), HBA (LifeSpan BioSciences; LS-B10549 1:100), PDGFRB (Abcam; ab32570; dilution, 1:100), and α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich; A2547; dilution, 1:50) to label smooth muscle cells or TF (R1073; dilution, 1:100) ( ).

Techniques: Immunofluorescence, Staining, Muscles, Comparison, Control

Journal: JCI Insight

Article Title: EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

doi: 10.1172/jci.insight.157701

Figure Lengend Snippet: ( A – D ) Representative western blot membranes and their quantification showing HBA, HBG2 (methemoglobin), and eNOS protein levels in murine primary endothelial cells isolated from lungs of EPCR fl/fl Tie2.Cre mice and EPCR fl/fl littermate controls; n = 3 biological replicates. ** P < 0.01 and **** P < 0.0001 versus EPCR fl/fl mice; unpaired Student’s t test. ( E and F ) Representative confocal images and the results of the quantitative analysis after visualization of HBA (red) expression in CD31 + endothelial cells (green) on cryopreserved cross sections isolated at day 28 after ischemia; n = 5 biological replicates. DAPI + cell nuclei appear blue. Scale bars: 10 μm. Note that myoglobin expression was not detected in endothelial cells in EPCR fl/fl Tie2.Cre mice and their littermate controls. ( G and H ) Representative confocal microscopy images and quantification of cryopreserved cross sections of nonischemic and ischemic hindlimbs of EPCR fl/fl Tie2.Cre mice and EPCR fl/fl littermate controls stained using antibodies against eNOS (red). DAPI + cell nuclei appear blue. Scale bars: 10 μm; n = 3 biological replicates per 2 experimental repeats; # P < 0.05 versus EPCR fl/fl control littermate mice using 2-way ANOVA, Sidak’s multiple-comparison test. ( I and J ) Representative microscopy brightfield images and quantification of total sprout length in control and DETA-NO–treated primary endothelial cell spheroids from EPCR fl/fl Tie2.Cre mice and EPCR fl/fl littermate controls; n = 3 biological replicates. *** P < 0.001 versus EPCR fl/fl littermate control mice; ### P < 0.001 versus untreated cells belonging to the same group; 1-way ANOVA, Sidak’s multiple-comparison test.

Article Snippet: Capillary density in the gastrocnemius muscle was assessed 28 days after induction of unilateral hindlimb ischemia on 5 μm–thick, acetone-fixed frozen sections (for 10 minutes at –20°C) stained with lycopersicon esculentum lectin or monoclonal rat antibodies against murine CD31 (Pecam1; Santa Cruz Biotechnology; clone MEC13.3; sc-18916; dilution, 1:50) to label immature, angiogenic endothelial cells , as well as antibodies against CD41 (Itga2b, Biozol; 11-763-C100; dilution, 1:100) to label platelets, eNOS (D9A5L; Cell Signaling Technology; 32027; dilution, 1:100), EPCR (R&D Systems; AF2245; dilution, 1:100), fibrinogen (Abcam; clone EPR18145-84; ab189490; dilution, 1:100), HBA (LifeSpan BioSciences; LS-B10549 1:100), PDGFRB (Abcam; ab32570; dilution, 1:100), and α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich; A2547; dilution, 1:50) to label smooth muscle cells or TF (R1073; dilution, 1:100) ( ).

Techniques: Western Blot, Isolation, Expressing, Confocal Microscopy, Staining, Control, Comparison, Microscopy

Journal: JCI Insight

Article Title: EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

doi: 10.1172/jci.insight.157701

Figure Lengend Snippet: ( A and B ) Representative confocal immunofluorescence microscopy images and quantitative analysis after visualization of HBA (red) expression in CD31 + endothelial cells (green) on cryopreserved cross sections isolated from mice at day 28 after ischemia. DAPI + cell nuclei appear blue. Scale bars: 10 μm. Two-way ANOVA, Sidak’s multiple comparison; n = 5 biological replicates. * P < 0.05 versus nonischemic hindlimb; ## P < 0.01 and ### P < 0.001 versus C57BL/6N control mice. ( C – E ) Representative Western blots and quantification of HBA and HBG2 (methemoglobin) protein levels in primary lung endothelial cells isolated from C57BL/6N, C57BL/6N PAR1 R41Q, and C57BL/6N PAR1 R46Q mutant mice; n = 3 biological replicates. Samples were run on the same blot. Nonadjacent lanes are indicated by an empty space between them. One-way ANOVA, Sidak’s multiple comparison. *** P < 0.001 and **** P < 0.0001 versus C57BL/6N; ## P < 0.01 and #### P < 0.0001 versus C57BL/6N PAR1 R41Q. Angiogenic sprout formation from aortic rings of C57BL/6N, C57BL/6N PAR1 R41Q, and C57BL/6N PAR1 R46Q mutant mice with and without DETA-NO (100 μM). ( F and G ) Representative findings and quantitative analysis are shown; n = 3 biological replicates. Scale bars: 100 μm. * P < 0.05 and ** P < 0.01 versus C57BL/6N; # P < 0.05 and ## P < 0.01 for DETA-NO versus untreated aortic rings of the same group. One-way ANOVA, Sidak’s multiple-comparison test.

Article Snippet: Capillary density in the gastrocnemius muscle was assessed 28 days after induction of unilateral hindlimb ischemia on 5 μm–thick, acetone-fixed frozen sections (for 10 minutes at –20°C) stained with lycopersicon esculentum lectin or monoclonal rat antibodies against murine CD31 (Pecam1; Santa Cruz Biotechnology; clone MEC13.3; sc-18916; dilution, 1:50) to label immature, angiogenic endothelial cells , as well as antibodies against CD41 (Itga2b, Biozol; 11-763-C100; dilution, 1:100) to label platelets, eNOS (D9A5L; Cell Signaling Technology; 32027; dilution, 1:100), EPCR (R&D Systems; AF2245; dilution, 1:100), fibrinogen (Abcam; clone EPR18145-84; ab189490; dilution, 1:100), HBA (LifeSpan BioSciences; LS-B10549 1:100), PDGFRB (Abcam; ab32570; dilution, 1:100), and α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich; A2547; dilution, 1:50) to label smooth muscle cells or TF (R1073; dilution, 1:100) ( ).

Techniques: Immunofluorescence, Microscopy, Expressing, Isolation, Comparison, Control, Western Blot, Mutagenesis

Journal: JCI Insight

Article Title: EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

doi: 10.1172/jci.insight.157701

Figure Lengend Snippet: ( A ) Representative IHC images of human skeletal muscle specimens from nonischemic controls (top panel) and patients with peripheral artery disease (PAD) showing expression of EPCR, HBA, and eNOS + endothelial cells; n = 4 biological replicates. Scale bars: 10 μm. ( B ) Representative IHC images of human PAD specimens showing expression of HBA, CD31, and EPCR on serial cross sections. Scale bars: 10 μm (upper row). Lower row shows enlarged areas of interest. Images are taken with 200× (upper row) and 400× (lower row) magnification. ( C ) Results of quantitative analysis of the number of positive cells residing in vascular specimens isolated from patients with PAD; n = 4 biological replicates. ** P < 0.01; 1-way ANOVA, Bonferroni’s multiple-comparison test. ( D ) Plasma levels of soluble EPCR (sEPCR) in 19 patients with PAD and 19 age- and sex-matched individuals with similar risk factors (RF) and no PAD diagnosis (controls with RF). ( E ) Plasma levels of nitrite/nitrate in patients with PAD and age-and sex-matched controls. * P < 0.05 and ** P < 0.01. Student’s t test.

Article Snippet: Capillary density in the gastrocnemius muscle was assessed 28 days after induction of unilateral hindlimb ischemia on 5 μm–thick, acetone-fixed frozen sections (for 10 minutes at –20°C) stained with lycopersicon esculentum lectin or monoclonal rat antibodies against murine CD31 (Pecam1; Santa Cruz Biotechnology; clone MEC13.3; sc-18916; dilution, 1:50) to label immature, angiogenic endothelial cells , as well as antibodies against CD41 (Itga2b, Biozol; 11-763-C100; dilution, 1:100) to label platelets, eNOS (D9A5L; Cell Signaling Technology; 32027; dilution, 1:100), EPCR (R&D Systems; AF2245; dilution, 1:100), fibrinogen (Abcam; clone EPR18145-84; ab189490; dilution, 1:100), HBA (LifeSpan BioSciences; LS-B10549 1:100), PDGFRB (Abcam; ab32570; dilution, 1:100), and α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich; A2547; dilution, 1:50) to label smooth muscle cells or TF (R1073; dilution, 1:100) ( ).

Techniques: Expressing, Isolation, Comparison, Clinical Proteomics, Biomarker Discovery

Journal: JCI Insight

Article Title: EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

doi: 10.1172/jci.insight.157701

Figure Lengend Snippet: ( A – C ) Human microvascular endothelial cells were treated with PTIO (100 μM), ODQ (50 μM), or the NO donor DETA-NO (100 μM), and changes in the mRNA transcript levels of platelet-endothelial cell adhesion molecule ( PECAM1 ) ( A ), smooth muscle α-actin ( ACTA2 ) ( B ), and platelet-derived growth factor receptor-β ( PDGFRB ) ( C ) were examined 4 days later using qPCR; n = 3-4 biological replicates. One-way ANOVA, Sidak’s multiple-comparison test. * P < 0.05 and *** P < 0.001 versus control; ## P < 0.01 and ### P < 0.001 versus ODQ-treated cells. ( D , E , H , and J ) Representative confocal images and quantitative analysis after immunostaining of SMA (red) and CD31 expression (green) in ischemic ( D and H ) and nonischemic hindlimb muscles of EPCR fl/fl Tie2.Cre mice and EPCR fl/fl littermate control mice or C57BL/6N, C57BL/6N PAR1 R41Q, and C57BL/6N PAR1 R46Q mutant mice ( E and J ); n = 5 biological replicates. DAPI + cell nuclei appear blue. Scale bars: 10 μm. ( F , G , I , and K ) Representative confocal images and ( I ) quantitative analysis after immunostaining of PDGFRB (red) and CD31 expression (green) in ischemic and nonischemic hindlimb muscles of EPCR fl/fl Tie2.Cre and EPCR fl/fl control littermate mice ( F and I ) or C57BL/6N, C57BL/6N PAR1 R41Q, and C57BL/6N PAR1 R46Q mutant mice at day 28 after ischemia ( G and K ); n = 5 biological replicates. DAPI + cell nuclei appear blue. Scale bars: 10 μm. * P < 0.05 and ** P < 0.01 versus nonischemic, contralateral hindlimb; ## P < 0.01, ### P < 0.001, and #### P < 0.0001 versus C57BL/6N control mice; §§ P < 0.01 versus C57BL/6N PAR1 R41Q mice. Two-way ANOVA, Sidak’s multiple-comparison test.

Article Snippet: Capillary density in the gastrocnemius muscle was assessed 28 days after induction of unilateral hindlimb ischemia on 5 μm–thick, acetone-fixed frozen sections (for 10 minutes at –20°C) stained with lycopersicon esculentum lectin or monoclonal rat antibodies against murine CD31 (Pecam1; Santa Cruz Biotechnology; clone MEC13.3; sc-18916; dilution, 1:50) to label immature, angiogenic endothelial cells , as well as antibodies against CD41 (Itga2b, Biozol; 11-763-C100; dilution, 1:100) to label platelets, eNOS (D9A5L; Cell Signaling Technology; 32027; dilution, 1:100), EPCR (R&D Systems; AF2245; dilution, 1:100), fibrinogen (Abcam; clone EPR18145-84; ab189490; dilution, 1:100), HBA (LifeSpan BioSciences; LS-B10549 1:100), PDGFRB (Abcam; ab32570; dilution, 1:100), and α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich; A2547; dilution, 1:50) to label smooth muscle cells or TF (R1073; dilution, 1:100) ( ).

Techniques: Derivative Assay, Comparison, Control, Immunostaining, Expressing, Muscles, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Structure matters: commensal Phocaeicola vulgatus lipopolysaccharide induces attenuated microglial activation and preserves neuronal integrity

doi: 10.3389/fncel.2026.1796397

Figure Lengend Snippet: Differential effects of E. coli and P. vulgatus LPS on BV2 microglial cells. (A) BV2 cell viability after stimulation with increasing concentrations of E. coli or P. vulgatus LPS (0.1–100 ng/mL) using the MTT assay. Unstimulated cells (NS) served as negative control. (B) Immunofluorescence analysis of microglial marker Iba-1 (red) to evaluate cell morphology and number following exposure to 100 ng/mL of LPS (scale bar = 100 μm). (C) The relative number of Iba 1 + cells from the experiments in (B) , normalized to NS (set at 100%). (D) Iba-1 immunofluorescence intensity was calculated as the mean area of the positive signal per cell. (E) Nitrite quantification and (F) western blot analysis of iNOS expression. Data are presented as mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. # p < 0.05; #### p < 0.0001 vs. NS; * p < 0.05; *** p < 0.001, **** p < 0.0001 vs. LPS.

Article Snippet: The murine BV2 microglial cell line (bladder carcinoma cell line-derived, viral oncogene-transformed; #305156) and the human microglial clone-3 cell line (HMC3; #300102) were purchased from Cytion Biosciences (Heidelberg, Germany).

Techniques: MTT Assay, Negative Control, Immunofluorescence, Marker, Western Blot, Expressing, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Structure matters: commensal Phocaeicola vulgatus lipopolysaccharide induces attenuated microglial activation and preserves neuronal integrity

doi: 10.3389/fncel.2026.1796397

Figure Lengend Snippet: Differential modulation of cytokine release and intracellular signaling in BV2 microglia. BV2 cells were treated with increasing concentrations (0.1, 1, 10, and 100 ng/mL) of LPS derived from E. coli or P. vulgatus . Unstimulated cells (NS) served as negative controls. (A) Cytokine levels (IL-1β, IL-6, and TNF-α) were quantified in the culture supernatants by using ELLA assay. (B) Activation of intracellular signaling pathways (STAT3, Nf-kb, ERK1/2) was assessed by western blotting. Data are presented as mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. # p < 0.05; ## p < 0.01; ### p < 0.001, #### p < 0.0001 vs. NS; * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001 vs. LPS.

Article Snippet: The murine BV2 microglial cell line (bladder carcinoma cell line-derived, viral oncogene-transformed; #305156) and the human microglial clone-3 cell line (HMC3; #300102) were purchased from Cytion Biosciences (Heidelberg, Germany).

Techniques: Derivative Assay, Activation Assay, Protein-Protein interactions, Western Blot, Comparison

Journal: Science Advances

Article Title: The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis

doi: 10.1126/sciadv.adg9278

Figure Lengend Snippet: ( A ) Western blotting for β-catenin in βC Control and βC ΔC MASMCs. Different antibodies targeting the C- or N-terminal domains (C-term or N-term, respectively) of β-catenin were used. * P < 0.05, unpaired Student’s t test. n = 3. ns, not significant. Data are means ± SEM of independent biological replicates. ( B ) Cell population growth of βC Control and βC ΔC MASMCs. * P < 0.05, two-way ANOVA with Šídák’s multiple comparison test. n = 6. Data are means ± SEM of independent biological replicates. ( C ) PCA from RNA-seq of βC Control and βC ΔC MASMCs. Each dot represents an independent sample. ( D ) Volcano plot. Each data point represents a gene. The log 2 fold change of each gene is represented on the x axis, and the log 10 of its adjusted P value ( P adj ) is on the y axis. Red dots indicate significantly up-regulated genes (log 2 fold change > 1 and P adj < 0.05), while blue dots represent significantly down-regulated genes (log 2 fold change < −1 and P adj < 0.05) in βC ΔC MASMCs compared to βC Control . The black dots represent genes that do not satisfy the above conditions. ( E ) Significantly enriched canonical signaling pathways identified by IPA and ranked by P value. Positive and negative z scores indicate up- and down-regulated signaling pathways, respectively, in βC ΔC versus βC Control MASMCs. ( F ) Heatmap derived from the RNA-seq data, showing the relative expression (color scale on the right) of Axin2 , a known β-catenin target gene, and key components of the S1P signaling pathway in βC Control and βC ΔC MASMCs. ( G ) qPCR for Axin2 , S1pr1 , and S1pr3 , which were significantly down-regulated in βC ΔC MASMCs in RNA-seq analysis. Rpl13 and β- actin were used as housekeeping controls. Statistical analysis: Unpaired Student’s t test. n = 6. Data are means ± SEM of independent biological replicates.

Article Snippet: To evaluate how the inhibition of β-catenin degradation affects S1PR1 expression, we treated βC Control and βC ΔC MASMCs with CHIR99021 (Cell Signaling Technology, 54290S) for 48 hours.

Techniques: Western Blot, Control, Comparison, RNA Sequencing, Protein-Protein interactions, Derivative Assay, Expressing

Journal: Science Advances

Article Title: The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis

doi: 10.1126/sciadv.adg9278

Figure Lengend Snippet: ( A ) Representative Western blotting and densitometric analysis of S1PR1 in βC Control and βC ΔC MASMCs, normalized to GAPDH. * P < 0.05, unpaired Student’s t test. n = 4. ( B ) Representative Western blotting and densitometric analysis of S1PR1 and β-catenin in βC Control and βC ΔC MASMCs electroporated with empty vector or β-catenin S33Y , a constitutively active form of β-catenin. * P < 0.05, two-way ANOVA with Šídák’s multiple comparison test. n = 3. ( C ) Representative Western blotting and densitometric analysis of S1PR1 and β-catenin in βC Control and βC ΔC MASMCs treated with CHIR99021 (CHIR), a small molecule that prevents β-catenin degradation. * P < 0.05, one-way ANOVA with Tukey’s multiple comparison test. n = 3. ( D ) βC Control and βC ΔC MASMCs were co-electroporated with a human S1PR1 -driven luciferase reporter (pGL3- S1PR1 -promoter) and empty vector or β-catenin S33Y . Forty-eight hours after transfection, cells were harvested for luciferase assay. Luminescence (lum) was normalized to β-galactosidase activity (β-gal) to control for transfection efficiency and expressed relative to empty vector in βC Control MASMCs. * P < 0.05, two-way ANOVA with Šídák’s multiple comparison test. N = 6. ( E ) CUT&RUN assay was conducted in MASMCs using anti–β-catenin, anti-TCF4, and anti-IgG antibodies and analyzed by qPCR using primers that amplify different regions in the S1pr1 promoter containing (+) or not (−) a consensus TCF binding motif, 5′-TCAAAG. Indicated locations are relative to transcription start site. * P < 0.05, unpaired Student’s t test. N = 4. ( F ) Cell population growth of βC Control , βC ΔC , βC Control S1PR1 GOF , and βC ΔC S1PR1 GOF MASMCs. * P < 0.05 compared to other groups, two-way ANOVA with Šídák’s multiple comparison test. n = 6. Data shown in (A) to (F) are means ± SEM of independent biological replicates.

Article Snippet: To evaluate how the inhibition of β-catenin degradation affects S1PR1 expression, we treated βC Control and βC ΔC MASMCs with CHIR99021 (Cell Signaling Technology, 54290S) for 48 hours.

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

Journal: Science Advances

Article Title: The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis

doi: 10.1126/sciadv.adg9278

Figure Lengend Snippet: ( A ) The β-catenin C-terminal inhibitor E7386 at 100 nM inhibits cell population growth of βC Control MASMCs but has no effect on βC ΔC MASMCs. Statistical analysis: Two-way ANOVA with Šídák’s multiple comparison test. n = 6. * P < 0.05 compared to other groups. Data are means ± SEM of independent biological replicates. ( B ) Model schematic—mouse carotid artery ligation was followed by gavage with vehicle or E7386 (50 mg/kg twice daily) before tissue analysis after 14 days. ( C ) E7386 reduces in vivo expression of Axin2, a typical target β-catenin signaling. Right panels show merge of Axin2 and SMA signals (yellow). Dotted line marks the IEL. ( D ) Percentage of SMA + cells positive for Axin2. n = 5 for each condition. ( E ) Representative images of H&E-stained carotid arteries at 14 days. Arrows delimit the neointimal growth. Scale bars, 50 μm. ( F to H ) Morphometric measurements of the medial area (F), intimal area (G), and a ratio of intimal to medial area (H). Each dot represents a single mouse; n = 7 for each group except n = 6 for E7386-treated females. Statistical analysis: Unpaired Student’s t test. Values are represented as means ± SEM of independent biological replicates. ( I ) E7386 reduces in vivo expression of S1PR1. Right panels show merge of S1PR1 and SMA signals (yellow). Dotted line marks the IEL. ( J ) Percentage of SMA + cells positive for S1PR1. n = 4 for each condition. Scale bar, 50 μm. Statistical analysis: Unpaired Student’s t test. Values are represented as means ± SEM of independent biological replicates.

Article Snippet: To evaluate how the inhibition of β-catenin degradation affects S1PR1 expression, we treated βC Control and βC ΔC MASMCs with CHIR99021 (Cell Signaling Technology, 54290S) for 48 hours.

Techniques: Control, Comparison, Ligation, In Vivo, Expressing, Staining

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: a Confocal images of frozen skeletal muscle sections from these four controls. We have replaced panel a with a correct version of these four controls, in which the control samples do have normal levels of PrP C (red). b , c H&E staining and immunohistochemical staining of the frozen sections showed that muscle regeneration was not observed in the skeletal muscle of these four controls. The experimental conditions are the same as those in Fig. . d Colocalization of PrP C and miR-214-3p in C2C12 mouse myoblasts (control) and C2C12 myoblasts stably expressing WT PrP C upon differentiation for 4 days. Shown are nuclei stained with DAPI (blue); signals were detected with the anti-PrP antibody 8H4 (red), and miR-214-3p was detected by FISH (green). Scale bar, 10 μm.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Staining, Immunohistochemistry, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: a Western blot for PrP C and the myogenic differentiation markers MyHC and MyoG during C2C12 mouse myoblasts (control), C2C12 myoblasts stably expressing full-length wild-type mouse PrP C (WT PrP C ), C2C12 myoblasts stably expressing F198S PrP C , and C2C12 myoblasts KO for PrP C cultured until their confluence reached 90% and then incubated with differentiation medium for 0, 3, and 5 days, respectively. β-actin served as the protein loading control. The relative amount of PrP C ( b ), MyHC ( c ), or MyoG ( d ) in the above cell lines (control, orange; WT PrP C , blue; F198S PrP C , gray; and KO for PrP C , pink) (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in n = 3 independent experiments. One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05. Immunofluorescence imaging of the above four cell lines incubated with differentiation medium for 3 and 5 days, respectively, using antibody against PrP C (red) ( e ) or MyHC (red) ( f ) and staining with DAPI (blue). Scale bars, 50 ( e ) and 75 μm ( f ), respectively. We have replaced the second row for Day 3 in ( f ) with a correct version of WT PrP C , in which WT PrP C -expressing cells did not have any MyHC signal at day 3 in most fields of view. Source data are provided as a file.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Western Blot, Stable Transfection, Expressing, Cell Culture, Incubation, Immunofluorescence, Imaging, Staining

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: Immunofluorescence imaging of C2C12 mouse myoblasts (control), C2C12 myoblasts stably overexpressing WT PrP C , and C2C12 myoblasts KO for PrP C cultured until their confluence reached 90% and then incubated with differentiation medium for 3 ( a ) and 5 ( b ) days, respectively, using antibodies against PrP C (red) and LC3B (green) and staining with DAPI (blue). The enlarged regions in the lower left corner ( a ) or the lower right corner ( b ) of the merged images show 4-fold enlarged images from the same images. Scale bars, 7.5 ( a ) and 50 ( b ) μm, respectively. We have replaced the first row in ( a ) with a correct version of control, in which LC3B did not overlap with PrP C in most fields of view. c Western blot for PrP C and the autophagy markers ATG5 and LC3B during the above three cell lines incubated with differentiation medium for 3 and 5 days, respectively. β-actin served as the protein loading control. The relative amount of ATG5 ( d ) or LC3B-II ( e ) in the above cell lines (control, orange; WT PrP C , blue; and KO for PrP C , gray) (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in n = 3 independent experiments. One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05. Source data are provided as a file.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Immunofluorescence, Imaging, Stable Transfection, Cell Culture, Incubation, Staining, Western Blot

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: a Profile of total miRNAs and PrP C IP-enriched miRNAs at day 4 of differentiation in C2C12 myoblasts stably overexpressing WT PrP C (WT PrP C ) and C2C12 myoblasts (control). Heat map shows the expression levels of miRNAs (high abundance, red; and low abundance, blue) and fold-enrichment compared to Inputs in the above two cell lines. b Western blotting analysis of PrP C immunoprecipitation in C2C12 myoblasts at day 4 of differentiation by probing PrP C and GAPDH. Volcano plot of 31 ( c ) and 51 ( d ) PrP C -bound miRNAs identified by small RNA-seq in differentiating C2C12 cells (control) and differentiating C2C12 cells stably expressing WT PrP C , respectively. Representative PrP C RIP-enriched miRNAs and depleted miRNAs are highlighted in orange and gray, respectively. Two-sided Student’s t -test was used to compare two sets of the small RNA-seq data. e Venn diagram of miRNAs enriched by PrP C RIP in C2C12 cells stably expressing WT PrP C versus miRNAs enriched by PrP C RIP in C2C12 myoblasts (control). Group I ( n = 21, Control RIP only, orange), n = 10 (overlap, brown), and group II ( n = 41, WT PrP RIP only, blue). RT−qPCR validation of depleted versus enriched miRNAs in differentiating C2C12 myoblasts (control) ( f ) and those stably expressing WT PrP C ( g ) normalized to total input. An axis break was introduced in the ordinate so that each orange bar is clearly paired with a gray bar as control (IgG). U6 snRNA served as a negative control. Data are presented as means ± SD ( n = 3 biologically independent measurements). The top six biological processes (BP, orange) of Gene Ontology (GO) enrichment in the control cells ( h ) and the top eight BPs of GO enrichment in C2C12 cells stably overexpressing WT PrP C ( i ). The differential genes annotated by GO terms were used to calculate the gene list and number of genes for each term. j Pulldown of endogenous PrP C and excess PrP C with WT and mutant biotin-labeled miR-214-3p or biotin-labeled miR-204-5p in the above two cell lines at day 4 of differentiation.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Stable Transfection, Expressing, Western Blot, Immunoprecipitation, RNA Sequencing Assay, Quantitative RT-PCR, Negative Control, Mutagenesis, Labeling

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: Dual luciferase reporter assays, in which miR-214-3p ( a ) or miR-204-5p ( b ) represses the wild-type 3’ end of untranslated region (WT 3’UTR) of the gene ATG5 or LC3B associated with luciferase. The relative luciferase activity of the reporters after transfection with NC (gray), miR-214-3p mimic ( a , orange), or miR-204-5p mimic ( b , blue) at day 4 of differentiation in C2C12 myoblasts stably overexpressing WT PrP C or KO for PrP C (solid black circles shown in scatter plots), and the relative stability of miR-214-3p ( c ), miR-204-5p ( d ), and nonspecific miR-183-5p ( e ) in C2C12 myoblasts stably overexpressing WT PrP C (red) or KO for PrP C (blue) incubated with differentiation medium for 4 days and then treated with 20 mg/ml α-amanitin, were expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. p = 0.001, 0.268, 0.000226, 0.467 ( a ), 0.035, 0.926, 0.001, 0.372 ( b ), 0.048 ( c ), 0.000086 ( d ), and 0.742 ( e ), respectively. Western blot for ATG5 ( f ), LC3B ( g ), and MyHC after transfection with NC, miR-214-3p inhibitor ( f ), or miR-204-5p inhibitor ( g ) at 10 μM in the aforementioned cells upon differentiation for 4 days. β-actin served as the protein loading control.The relative amount of ATG5 ( h ), LC3B-II ( j ), or MyHC ( i , k ) in the aforementioned cells (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Transfection of NC, miR-214-3p inhibitor ( h , i ), or miR-204-5p inhibitor ( j , k ) in C2C12 myoblasts stably overexpressing WT PrP C (orange or blue) or KO for PrP C (gray). a – e Statistical analyses were performed using two-sided Student’s t -test. Values of p < 0.05 indicate statistically significant differences. The following notation is used throughout: * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001 relative to controls. n.s., no significance. h – k One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Luciferase, Activity Assay, Transfection, Stable Transfection, Incubation, Western Blot

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: a Schematic of the optoIDR assay, depicting recombinant protein with an IDR (blue), mCherry (red), and Cry2 (orange) expressed in cells exposed to 488-nm laser. b – e Time-lapse images of living C2C12 cells expressing mCherry-Cry2-WT PrP C construct containing PrP 1-37 IDR (blue) linked to mCherry (red), Cry2 (orange), and PrP 38-230 IDR (blue) ( c – e ), and mCherry-Cry2 fusion alone as a control ( b ). A droplet fusion event occurs in the region (orange box, ( d )); and orange arrows, ( e ). Cells were subjected to laser excitation every 2 s for the indicated time. f Representative live-cell images of PrP C -miRNA speck formation when FAM-labeled miRNA (green) was transfected into C2C12 cells expressing mCherry or WT PrP C -mCherry (red), among ≥ 10 cells. FRAP analysis on the selected liquid droplets of PrP C (red) in living C2C12 cells expressing mCherry-Cry2-WT PrP C transfected without ( g ) or with ( h ) miRNA before (prebleach), during (0 s), and after photobleaching (60 and 125 s). Dashed white circle highlights the punctum undergoing targeted bleaching. Scale bars, 7.5 μm ( b – h ). Quantification of FRAP data ( i ) and normalized fluorescence intensity ( j ) of WT PrP C puncta (blue circle) and WT PrP C puncta + miR-214-3p (red square) were expressed as the mean ± SD (with error bars) of values ( n = 3). The solid lines show the best single exponential fit ( j ). k Time-lapse images of living C2C12 cells expressing mCherry-Cry2-WT PrP C treated with 2.5% 1,6-hexanediol. Scale bar, 5 μm. l Western blot for ATG5 and LC3B in C2C12 cells expressing mCherry-Cry2-WT PrP C transfected without (-) or with (+) miRNA and then treated with (+) or without (-) 1,6-hexanediol. The relative amount of ATG5 ( m ) and LC3B-II ( n ) in the aforementioned cells (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Recombinant, Expressing, Construct, Labeling, Transfection, Fluorescence, Western Blot

Journal: Nature Communications

Article Title: Excess PrP C inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

doi: 10.1038/s41467-023-43826-7

Figure Lengend Snippet: a Western blot for PrP in the sarkosyl-insoluble pellets and the corresponding cell lysates from C2C12 cells stably overexpressing mCherry-Cry2-WT PrP C transfected without (-) or with (+) 10 μM miR-214-3p and cultured for 12 h after activation by 488-nm laser for 10 min. β-actin served as the protein loading control. b The normalized amount of insoluble PrP aggregates in the aforementioned cells (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. c The cell viability (%) (solid black circles shown in scatter plots) was measured by MTT reduction assay and expressed as the mean ± SD (with error bars) of values obtained in five independent experiments. C2C12 cells expressing mCherry-Cry2 (gray) or expressing mCherry-Cry2-WT PrP C transfected without (-) (orange) or with (+) (blue) 10 μM miR-214-3p ( b , c ). Immunogold electron microscopy of PrP fibrils purified from the same C2C12 myoblasts stably expressing mCherry-Cry2-WT PrP C transfected without ( d ) or with ( e ) 10 μM miR-214-3p as in ( a ), and labeled by gold particles conjugated with anti-PrP antibody. Scale bars, 100 nm. f Western blot for PrP in the sarkosyl-insoluble pellets and the corresponding cell lysates from C2C12 myoblasts stably overexpressing WT PrP C incubated with differentiation medium for 2 days, transfected without or with 10 or 20 μM miR-214-3p and cultured for 48 h. β-actin served as the protein loading control. g the same as in ( b ). C2C12 cells expressing WT PrP C transfected without or with 10 (gray) or 20 μM (orange) miR-214-3p. b , c , g One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05. h Western blot for PrP in the cell lysates from the same C2C12 cells stably overexpressing mCherry-Cry2-WT PrP C transfected without or with 10 μM miR-214-3p as in ( a ), and digested with 0.25, 0.50, 0.75, 1.0, and 2.0 μg/ml proteinase K.

Article Snippet: Murine-derived C2C12 myoblast cells (catalog number GDC0175) and HEK-293T cells (catalog number GDC0187) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Western Blot, Stable Transfection, Transfection, Cell Culture, Activation Assay, MTT Reduction Assay, Expressing, Electron Microscopy, Purification, Labeling, Incubation