Journal: The Journal of Cell Biology

Article Title: IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs

doi: 10.1083/jcb.201410052

Figure Lengend Snippet: IL-1α–induced atypical apoptosis with caspase-3 activation in fetal liver MKs. (A and B) Liver cells were obtained from fetal WT mice on embryonic day 13, cultured with TPO or IL-1α, and analyzed on day 7. (A) RT-PCR analysis of gene expression in the harvested cells. The values were normalized to a vehicle-treated control. n = 5 experiments. (B) Flow cytometric analysis of caspase-3 activation in Lin − CD41 + CD42b + MKs. (C) Fetal liver cells were cultured with TPO. siRNA-mediated knockdown was performed on day 4. On day 7, cells were washed, incubated with TPO, IL-1α, or IL-1β for an additional 1 d, and pAKT–pERK signaling in Lin − CD41 + CD42b + MKs was analyzed on day 8. The data shown are from a single representative experiment from among three repeats (B and C). (D) Western blotting of p53 and phospho-p53 in fetal liver cells cultured with TPO and IL-1α from day 0 to 7. MKs were enriched with discontinuous albumin density gradient centrifugation. (E) Fetal liver cells were differentiated using TPO for 7 d, and then stimulated with vehicle, TPO, IL-1α, or IL-1β for an additional 1 d, after which gene expression was analyzed using RT-PCR. (F) Flow cytometric analysis of T. orange high and T. orange low platelets isolated from WT mice treated with TPO or IL-1α. NC denotes a negative control. The data shown are from a single representative experiment from among five repeats. (G–J) Immunofluorescence analysis of fetal liver MKs, which were cultured and differentiated from day 0 to 7 with TPO or IL-1α. Some cells were treated with Fas-ligand from day 6 to 7 after differentiation with TPO (FasL). Note that IL-1α–treated MKs were caspase-3–positive with release of von Willebrand factor–positive granules, but TUNEL staining was negative, which was different than typical FasL-induced apoptosis with blebbing. (K) Blood cell counts in WT mice treated with TPO, IL-1α, and/or Z-VAD (OMe)-FMK. (L) MK dynamics and platelet counts in WT, Casp3 −/− , and Thpo −/− mice treated with vehicle or IL-1α. n = 3–8 mice. Bars, (red) 20 µm. *, P < 0.05 versus CTRL group.

Article Snippet: Caspase activation was evaluated using NucView Caspase-3 (Biotium; ).

Techniques: Activation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Western Blot, Gradient Centrifugation, Isolation, Negative Control, Immunofluorescence, TUNEL Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

doi: 10.1111/jcmm.17280

Figure Lengend Snippet: LRG‐1 mitigated the apoptosis of the transplanted fat. (A) Images of H&E‐stained sections of the PBS injection and LRG‐1 injection groups. (Scale bar = 200/100 μm). (B) Images and quantitative analysis of immunofluorescence staining of Perilipin (green). (DAPI (blue) for nuclei staining, Scale bar = 100 μm). (C) Images and quantitative analysis of immunohistochemistry staining of cleaved caspase‐3. (Scale bar = 200/100 μm). (D) Apoptosis was detected in fat grafts in the PBS‐ and LRG‐1‐injected groups by flow cytometry. (E) Western blot analysis of Bax, Bcl‐2 and cleaved caspase‐3 in fat grafts in different groups. Data are presented as the mean ± SD. ( n = 8 biologically independent animals) * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The separated proteins were then immunoblotted and probed with primary anti‐Bax antibody (Abcam, ab32503, 1:5000), antiBcl‐2 antibody (Abcam, ab182858, 1:2000), anti‐Cleaved caspase 3 antibody (Abcam, ab208003, 1:1000), antiRAB31 antibody (Abcam, ab230881, 1:1000), antiβ‐actin antibody (Abcam, ab8227, 1:5000), antiMLKL (Abcam, ab184718, 1:1000), antip‐MLKL (Abcam, ab196436, 1:1000), antiRIPK1 (CST, 3493T, 1:1000), antip‐RIPK1 (proteintech, 66854‐1‐Ig, 1:500), antiRIPK3 (proteintech, 17563–1‐AP, 1:500), antip‐RIPK3 (Abcam, ab184718, 1:500), antiLc3b (Abcam, ab192890, 1:1000), antip62 (Abcam, ab109012, 1:1000), antiATGL (Abcam, ab207799, 1:1000) and antiHSL (Abcam, ab109400, 1:50000) at 4°C overnight.

Techniques: Staining, Injection, Immunofluorescence, Immunohistochemistry, Flow Cytometry, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

doi: 10.1111/jcmm.17280

Figure Lengend Snippet: LRG‐1 reduced hypoxia‐induced h‐ADSC apoptosis. (A) Apoptosis was detected after treating h‐ADSC with PBS or LRG‐1 for 48 h under normal oxygen (21% O 2) culture conditions by flow cytometry. (B) Western blot analysis of Bax, Bcl‐2 and cleaved caspase‐3 in h‐ADSC after treatment with PBS or LRG‐1 (300 ng/ml or 500 ng/ml) for 48 h under normal oxygen (21% O 2 ). (C) Apoptosis was detected after treating h‐ADSC with PBS or LRG‐1 for 48 h under hypoxic (1% O 2 ) culture conditions by flow cytometry. (D) Western blot analysis of Bax, Bcl‐2 and cleaved caspase‐3 in h‐ADSC after treatment with PBS or LRG‐1 for 48 h under hypoxia (1% O 2 ). Data are presented as the mean ± SEM. ( n = 3 independent experiments) * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The separated proteins were then immunoblotted and probed with primary anti‐Bax antibody (Abcam, ab32503, 1:5000), antiBcl‐2 antibody (Abcam, ab182858, 1:2000), anti‐Cleaved caspase 3 antibody (Abcam, ab208003, 1:1000), antiRAB31 antibody (Abcam, ab230881, 1:1000), antiβ‐actin antibody (Abcam, ab8227, 1:5000), antiMLKL (Abcam, ab184718, 1:1000), antip‐MLKL (Abcam, ab196436, 1:1000), antiRIPK1 (CST, 3493T, 1:1000), antip‐RIPK1 (proteintech, 66854‐1‐Ig, 1:500), antiRIPK3 (proteintech, 17563–1‐AP, 1:500), antip‐RIPK3 (Abcam, ab184718, 1:500), antiLc3b (Abcam, ab192890, 1:1000), antip62 (Abcam, ab109012, 1:1000), antiATGL (Abcam, ab207799, 1:1000) and antiHSL (Abcam, ab109400, 1:50000) at 4°C overnight.

Techniques: Flow Cytometry, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

doi: 10.1111/jcmm.17280

Figure Lengend Snippet: RAB31 mediated the inhibitory effect of LRG‐1 on apoptosis in h‐ADSC under hypoxic conditions. (A) Top 10 upregulated and downregulated genes from transcriptome data analysis of the control group and LRG‐1 additional group ( GSE122527 ). (B) The levels of RAB31 mRNA in h‐ADSC treated with PBS or LRG‐1 for 48 h under normal oxygen (21% O2) or hypoxia (1% O2). (C) The levels of RAB31 protein in h‐ADSC treated with PBS or LRG‐1 for 48 h under hypoxia (1% O2). (D) Apoptosis was detected after treating h‐ADSC with PBS, LRG‐1, LRG‐1 with siNC and LRG‐1 with siRAB31 for 48 h under hypoxia (1% O2). (E) The protein levels of Bax, Bcl‐2 and cleaved caspase‐3 were detected in the different groups. Data are presented as the mean ± SEM. ( n = 3 independent experiments) * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The separated proteins were then immunoblotted and probed with primary anti‐Bax antibody (Abcam, ab32503, 1:5000), antiBcl‐2 antibody (Abcam, ab182858, 1:2000), anti‐Cleaved caspase 3 antibody (Abcam, ab208003, 1:1000), antiRAB31 antibody (Abcam, ab230881, 1:1000), antiβ‐actin antibody (Abcam, ab8227, 1:5000), antiMLKL (Abcam, ab184718, 1:1000), antip‐MLKL (Abcam, ab196436, 1:1000), antiRIPK1 (CST, 3493T, 1:1000), antip‐RIPK1 (proteintech, 66854‐1‐Ig, 1:500), antiRIPK3 (proteintech, 17563–1‐AP, 1:500), antip‐RIPK3 (Abcam, ab184718, 1:500), antiLc3b (Abcam, ab192890, 1:1000), antip62 (Abcam, ab109012, 1:1000), antiATGL (Abcam, ab207799, 1:1000) and antiHSL (Abcam, ab109400, 1:50000) at 4°C overnight.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

doi: 10.1111/jcmm.17280

Figure Lengend Snippet: RAB31 mediated the inhibitory effect of LRG‐1 on apoptosis of fat grafts. (A) Images of H&E‐stained sections in the control, LRG‐1 injection, LRG‐1 combined with AAV‐shCtrl injection and LRG‐1 combined with AAV‐shRAB31 injection groups (scale bar = 200/100 μm). (B) Images and quantitative analysis of immunofluorescence staining of Perilipin (green). (DAPI (blue) for nuclei staining, Scale bar =100 μm). (C) Images and quantitative analysis of immunohistochemistry staining of cleaved caspase‐3. (Scale bar = 200/100 μm). (D) Apoptosis was detected in fat grafts in the control, LRG‐1 injection, LRG‐1 combined with AAV‐shCtrl injection and LRG‐1 combined with AAV‐shRAB31 injection groups by flow cytometry. (E) Western blot analysis of Bax, Bcl‐2 and cleaved caspase‐3 in fat grafts in different groups. Data are presented as the mean ± SD. ( n = 8 biologically independent animals) * p < 0.05, *** p < 0.001

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The separated proteins were then immunoblotted and probed with primary anti‐Bax antibody (Abcam, ab32503, 1:5000), antiBcl‐2 antibody (Abcam, ab182858, 1:2000), anti‐Cleaved caspase 3 antibody (Abcam, ab208003, 1:1000), antiRAB31 antibody (Abcam, ab230881, 1:1000), antiβ‐actin antibody (Abcam, ab8227, 1:5000), antiMLKL (Abcam, ab184718, 1:1000), antip‐MLKL (Abcam, ab196436, 1:1000), antiRIPK1 (CST, 3493T, 1:1000), antip‐RIPK1 (proteintech, 66854‐1‐Ig, 1:500), antiRIPK3 (proteintech, 17563–1‐AP, 1:500), antip‐RIPK3 (Abcam, ab184718, 1:500), antiLc3b (Abcam, ab192890, 1:1000), antip62 (Abcam, ab109012, 1:1000), antiATGL (Abcam, ab207799, 1:1000) and antiHSL (Abcam, ab109400, 1:50000) at 4°C overnight.

Techniques: Staining, Injection, Immunofluorescence, Immunohistochemistry, Flow Cytometry, Western Blot

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: E6AP autism mouse model neurons show impairment in spine maturation and reduction in dendritic branching. A, Brain lysates collected from WT or 2X Tg mice at P15 were probed for E6AP, XIAP, caspase-3, cleaved caspase-3, cleaved tubulin, and total tubulin. GAPDH was also probed as a loading control. B–D, Quantification analysis of Western blots for XIAP, cleaved caspase-3, and cleaved tubulin; n = 3 for each. E, At P15, brains of WT and 2X Tg mice were subjected to Golgi staining. Representative images of spine morphology of layer-V somatosensory cortical neurons are shown. F, Mean spine density decreased in 2X Tg mice; n = 10 neurons. G, Mean spine length increased in 2X Tg mice; n = 10 neurons. H, I, The percentage and number of filopodia increased in 2X Tg mice; n = 10 neurons. J, Representative layer-V pyramidal neuron tracing images of Golgi staining from P15 WT and 2X Tg mouse brain slices. K, L, Measurement of average dendrite number and total dendritic length in pyramidal neurons; n = 12 neurons. Error bars represent SEM, *p < 0.05, **p < 0.01 (Fig. 7-1, ). Summary of the E6AP-dependent dendritic remodeling pathway.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Western Blot, Staining

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: Activation of caspase-3 is required for E6AP-dependent dendritic remodeling. A, Neurons were transfected with surfGFP (green; Control) or together with E6AP, and the cleaved (activated) caspase-3 (red) was immunostained 24 h later. Scale bar, 50 μm. B, Quantification of the cleaved caspase-3 immunofluorescence signals; n = 10. E6AP expression resulted in higher levels of cleaved caspase-3. C, D, DIV 2 hippocampal neurons were infected with AAV9 GFP virus or AAV9 E6AP virus for 10 d and cleaved caspase-3 levels were measured by Western blot. Neurons infected with E6AP virus showed higher levels of cleaved caspase-3; n = 3 independent experiments. E, Dendritic arborization reduction in E6AP neurons was blocked by inhibiting caspase-3 cleavage with the caspase-9 inhibitor Ac-LEHD-CMK (150 nm) at the time of transfection, as shown by Sholl analysis; n = 10. F, G, Neurons were transfected with surfGFP (control) or together with E6AP or E6AP + Casp3 C163A (E6AP + C3 mutant), a catalytic caspase-3 mutant. Scale bar, 50 μm. Sholl analysis revealed a rescue of the E6AP-induced dendritic remodeling by Casp3 C163A; n = 10. Error bars represent SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Activation Assay, Transfection, Immunofluorescence, Expressing, Infection, Western Blot, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: Microtubule cleavage and retraction in E6AP-induced dendritic remodeling. A, Neurons were transfected with surfGFP and pTRE-E6AP for 24 h, and loaded with SiR-Tubulin, a fluorogenic and cell-permeable dye for tubulin labeling, before being treated with doxycycline (Dox) to induce E6AP expression. Tubulin and surfGFP images were obtained every 20 min for 12 h following Dox application. Representative images show that retraction of microtubule (red; hollow arrowhead) occurred before that of the GFP-positive dendritic branch (green; solid arrowhead). The original position of the dendritic tip is indicated by a dashed line. Scale bar, 5 μm. B, Neurons were infected with AAV9 GFP virus or AAV9 E6AP virus for 10 d, and cleaved tubulin levels were measured by Western blot with an antibody specifically against the cleaved microtubule (ΔTubulin). C, Quantification showed an increased level of microtubule cleavage in E6AP-infected neurons; n = 3 independent experiments. D, Representative image of E6AP neurons immunostained with ΔTubulin. Scale bar, 10 μm. E, Quantification of ΔTubulin immunointensity in neurons transfected with vector control, E6AP, or E6AP + Casp3 C163A (E6AP + C3 mutant), compared with control; n = 10. F, Morphology of neurons transfected with surfGFP, tubulin WT, E6AP, or E6AP + tubulin WT. Scale bar, 50 μm. G, Sholl analysis of dendritic arborization; n = 10 cells per condition. H, Quantification of total dendritic length; n = 10 cells per condition. Changes in tubulin stabilization also affected the E6AP-dependent dendritic remodeling (Fig. 5-1, . Error bars represent SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Transfection, Labeling, Expressing, Infection, Western Blot, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Violin plots depict limb muscle single cell RNAseq data for Dlk1 - Dio3 megacluster-encoded lncRNAs Meg3 , Rian , and Mirg . All lncRNAs showed enrichment in satellite (sat.) and mesenchymal (mes.) cell types, with Meg3 as the most abundant. CPM = counts per million reads mapped. B) qPCR temporal Meg3 expression profiling was performed on regenerating mouse Tibialis anterior (TA) muscle tissue harvested before (uninjured), and on the indicated days after cardiotoxin injection (+CTX). Meg3 lncRNA transcripts were upregulated following CTX-induced injury, which corresponds with satellite and mesenchymal cell expansion (n=3 mice per time point). C) qPCR temporal expression profiling of Meg3 in C2C2 myoblast differentiation. Meg3 transcripts were most enriched during proliferation (prolif.), and progressively downregulated during course of differentiation (n=4). D) RNA immunoprecipitation (RNA-IP) was performed on subconfluent C2C12 myoblast lysates to examine for Meg3 -PRC2 interaction. Immunoprecipitated RNA was quantified by qPCR, using supernatant as an internal normalization control. Compared to normal IgG controls, Meg3 was enriched in anti-Ezh2 immunoprecipitates (n=3 sets of 60 plates).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Expressing, Injection, RNA Immunoprecipitation, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) qPCR quantification of Meg3 transcript levels in heterogeneous cell populations derived from G418 selection (C2C12 het.), and subsequently derived clonal populations (C2C12 clones) indicate that stable shRNA integration resulted in Meg3 knockdown (n=3). B) Immunofluorescence quantification of MYH4 indicates markedly reduced expression in sh Meg3 C2C12 clones. Quantification of nuclei within α-actinin cell-boundaries show reduced fusion index in sh Meg3 clones (n=3). qPCR expression profiling indicated unchanged Myf5 transcript levels, but significant reduction in other myogenic differentiation markers (n=3). C) Western blot quantification of MF20 signal (normalized to β-tubulin) showed marked reduction specific to sh Meg3 clones (n=4). D) qPCR quantification confirmed overexpression of human MEG3 in sh LacZ and sh Meg3 myoblasts, and restoration of endogenous Meg3 transcript levels relative to β-galactosidease controls (n=3). E) Human MEG3 restored both MYH4 expression and fusion index in sh Meg3 but not sh LacZ myoblasts (n=6 MYH4, n=3 fusion index). F) qPCR expression profiling of heterogeneous rescue clones revealed an increase in Mef2C, Ckm , MyoD and Myog levels in sh Meg3 + MEG3 myotubes. MYH4 = myosin heavy chain 4 (Proteintech antibody), MF20 = myosin heavy chain 4 (DHSB antibody), Myf5 = myogenic factor 5 , MyoD = myogenic differentiation 1, Mef2C = myogenic enhancing factor- 2 C , Myog = Myogenin , Ckm = Muscle Creatine Kinase , Acta1 = skeletal muscle actin.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Derivative Assay, Selection, Clone Assay, shRNA, Knockdown, Immunofluorescence, Expressing, Western Blot, Over Expression

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Quantification of BrdU+ nuclei (green arrowheads) indicated that sh Meg3 myoblasts divided at a reduced frequency (n=3). B) Cleaved caspase 3 assay revealed higher apoptosis in sh Meg3 myoblasts and myotubes relative to control (n=3). Cell Titer Blue viability assay indicated reduced viability in sh Meg3 myoblasts and myotubes relative to control (n=3). C) sh Meg3 myoblasts seeded at increasing densities was not sufficient to restore MYH4 expression or fusion index (n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Caspase-3 Assay, Control, Viability Assay, Expressing

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: C2C12 myoblasts and myotubes were pulsed with MitoTracker CMXRos for 40 minutes, and co-stained with α-actinin. Quantification of Mitotracker (restricted to α-actinin+ cells) indicated reduced mitochondrial signal in sh Meg3 myoblasts, but not myotubes (n=3). Both treatment groups displayed increased MitoTracker signal with differentiation.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Staining

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: RNAseq data indicated that transcripts of master regulators of myogenic fusion, notably Myomaker and Myomixer , were not significantly downregulated in sh Meg3 myotubes.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques:

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Cadherin switching was assessed by qPCR (left) and Western blot (right) quantification of E-cadherin (Cdh1) and N-cadherin (Cdh2) in day 3 myotubes. Cdh1 transcripts were significantly downregulated in sh Meg3 myotubes relative to sh LacZ controls (n=3), but Cdh1 protein signal from myotubes lysates was extremely faint (n=4). Cdh2 transcripts were upregulated (n=3), as was Cdh2 protein (n=4). B) qPCR expression profiling indicated significant downregulation of epithelial Plakophilin and PatJ transcripts in sh Meg3 myotubes, with simultaneous upregulation of mesenchymal Fibronectin and Snai2 (n=3). C) Western blot of Vimentin normalized to β-tubulin indicated no change in mesenchymal Vimentin (n=4). D) Scratch-wound assays revealed no detectable change in wound-healing efficiency, as measured by % scratch area (n=3). Brightfield (BF) microscopy of wound-healing morphology differed between sh LacZ control and sh Meg3 clones, with a higher proportion of myoblasts invading the scratch territory with fewer than 2 cell contacts (black arrowheads, n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Western Blot, Expressing, Microscopy, Control, Clone Assay

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Following incubation with 10μM LY2157299 (LY), myoblasts were induced to differentiate and examined for MYH4 expression and fusion index. LY-treated sh Meg3 myotubes adopted an elongated morphology, and displayed increased MYH4, decreased myotubes with 1-nuclei, and increased myotubes with 2- and ≥3 nuclei relative to untreated sh Meg3 controls (n=3). B) Western blot for MF20 indicated that LY-treatment restored myosin heavy chain 4 expression to sh Meg3 myotubes (n=3). C) qPCR expression profiling of LY-treated myotubes indicate significant upregulation of all myogenic markers surveyed ( Myf5 , MyoD , Mef2C , Myog , Ckm , Acta1 ) relative to untreated sh LacZ myotubes.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Incubation, Expressing, Western Blot

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) qPCR indicated that LY2157299 (LY) treatment resulted in reduced E-cadherin ( Cdh1 ) transcripts regardless of sh RNA treatment, with simultaneous upregulation of N-cadherin ( Cdh2 ) transcripts (n=3). Western blot revealed modest Cdh1 band detection, and quantification of β-tubulin-normalized signal revealed that LY-treatment restored Cdh1 levels to sh Meg3 myotubes, while Cdh1 in sh LacZ myotubes remained unchanged. While LY treatment did not change Cdh2 expression in sh LacZ myotubes, LY-treatment enhanced Cdh2 signal in sh Meg3 myotubes. B) qPCR profiling indicated upregulation of epithelial transcripts Plakophilin and PatJ regardless of sh RNA background (n=3). Fibronectin transcript levels returned to normal levels in LY-treated sh Meg3 myotubes. LY treatment intensified upregulation of Snai2 transcripts in sh Meg3 cells, but did not affect Twist2 or Mmp9 levels relative to sh Meg3 myotubes. sh LacZ + LY myotubes displayed reduced Mmp9 , with simultaneous upregulation of Twist2 when compared to untreated sh LacZ cells (n=3). C) Western blot quantification of Vimentin suggests that LY treatment reduced Vimentin expression in sh LacZ controls, but did not change Vimentin expression in sh Meg3 myotubes (n=3). D) Myoblasts pre-treated with 5ng/mL BMP4 (BMP) were subjected to differentiation, and examined for changes in MYH4 expression and fusion index. BMP4 treated sh Meg3 myotubes had improved MYH4 expression (n=3), reduced mononucleated myotubes, and improved 2-cell fusion, but not ≥3 nuclei fusion.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Following incubation with 40μM ROCK1/2 inhibitor (Y-27632), myoblasts were induced to differentiate and examined for MYH4 expression and fusion index. Y-27632-treated sh Meg3 myotubes adopted an elongated morphology, and fusion quantification indicated decreased myotubes with 1-nuclei, and increased myotubes with 2- and ≥3 nuclei relative to sh Meg3 control (n=3). While MYH4 expression was enhanced in sh LacZ + Y-27631 myotubes, MYH4 levels remained unchanged with Y-27632 treatment in sh Meg3 cells (n=3). B) Following incubation with 5μM p38 inhibitor (SB203580), myoblasts were induced to differentiate and examined for MYH4 expression and fusion index. SB203580-treated sh Meg3 myotubes adopted an elongated spindle-like morphology, and fusion quantification indicated decreased myotubes with 1-nuclei, and increased myotubes with 2- and ≥3 nuclei relative to sh Meg3 control (n=3). MYH4 expression was unaffected by SB203580 treatment (n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Incubation, Expressing, Control

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) qPCR expression profiling indicated reduced Meg3 expression in TA muscles co-injected with sh Meg3 adenovirus (n=3). B) Whole mount morphology of regenerating muscles co-injected with adeno-sh LacZ (top) or adeno-sh Meg3 . C) Hematoxylin and eosin (H&E) staining of muscle sections. D) Cross-sectional area (CSA) of laminin-ensheathed regenerating myofibers was measured for days 3, 7, and 14 post-CTX injury. sh Meg3 muscle displayed reduced CSA for all time points surveyed. E) Immunofluorescence quantification indicated sh Meg3 TA sections harbor increased Ki67 signal (left bar graph). Co-staining for Pax7 indicated no change in satellite-cell specific Ki67 signal (left graph) and no change in satellite cell abundance (white, red arrowheads). Marker quantification revealed an increase in proliferating cells lacking Pax7 co-stain (green arrowheads). F ) Immunofluorescence quantification indicated an increase in PDGFR signal, as well as increased abundance of PDGFRα+ cells (red arrowheads).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Expressing, Muscles, Injection, Staining, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Expression profiling by qPCR indicated no change in E-cadherin , while day 7 sh Meg3 muscle was enriched for N-cadherin transcripts (n=3). Immunofluorescence revealed that, while numerous satellite cells were not N-cad+ (red arrowheads), N-cad signal was largely restricted to Pax7+ satellite cells in regenerating muscle (white arrowheads). Cell quantification (% mononuc. cells) revealed that sh Meg3 muscle harbored increased abundance of N-cadherin+ satellite cells, whereas levels of N-cadherin per cell was unchanged (n=3). B) qPCR expression profiling indicated no change in epithelial markers Plakophilin and PatJ , but mesenchymal markers Fibronectin and Snai2 were significantly upregulated in sh Meg3 muscle (n=3). C) Immunofluorescence revealed the presence of satellite cells lacking Snai2 (red arrowheads), Snai2+ satellite cells (white arrowheads), Snai2+ non-satellite cells (green arrowheads), and Snai2+ nuclei in regenerating myofibers (green asterisks). Quantification of Snai2+ cells indicated no change in the occurance of Snai2+ nuclei (bar graph, % Snai2+ nuclei). Generalized analysis (DAPI) indicated significant upregulation of cytoplasmic Snai2 signal per cell, but no change nuclear intensity; Snai2 signal in satellite cells (Pax7+) was increased for both cytoplasmic and nuclear compartments (n=3). D) Immunofluorescence revealed the presence of satellite cells lacking Vimentin (red arrowheads), Vimentin+ satellite cells (white arrowheads), and Vimentin+ non-satellite cells (green arrowheads). Cell quantification (% mononuc. cells) revealed increased abundance of Vimentin+ mononucleated cells that were not Pax7+, and that Pax7+/Vimentin+ cells were reduced in sh Meg3 muscle. Vimentin signal per cell was downregulated in mononucleated cells (bottom right bar graph), which may reflect sh Meg3 -specifici differences in Vimentin+ cell morphology (green channel) (n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Expressing, Immunofluorescence