Journal: bioRxiv

Article Title: Targeting de novo cholesterol synthesis in rhabdomyosarcoma induces cell cycle arrest and triggers apoptosis through ER stress-mediated pathways

doi: 10.1101/2024.12.18.628900

Figure Lengend Snippet: Cholesterol biosynthesis is elevated in rhabdomyosarcoma (RMS). (A) Venn diagram illustrating the number of significantly downregulated genes identified through RNA-seq analysis following PROX1 silencing in RMS cell lines RD and KLHEL1 and compared to healthy human myoblasts. (B) Gene ontology analysis of the 436 common genes downregulated upon PROX1 silencing in both RD and KLHEL1 cells but not in human myoblasts. (C) Gene Set Enrichment Analysis (GSEA) demonstrates the consistent repression of genes involved in cholesterol biosynthesis pathway upon PROX1 silencing in RD cells (left) and in KLHEL1 cells (right). NES denotes normalized enrichment score, and FDR indicates false-discovery rate. (D) Quantification of total cellular cholesterol content in shSCR and shPROX1 treated RD cells (left) and KLHEL1 cells (right) at 3 days post-transduction. The data were analyzed following organic extraction and normalization to protein levels. Data presented as mean ± standard error of the mean (SEM) (*p < 0.05). (E) Normalized expression (FPKM) of genes involved in cholesterol biosynthesis in normal muscles, fusion-negative RMS (FN-RMS), and fusion-positive RMS (FP-RMS) tumor samples in analyzed using a publicly available RNA-seq data set (GSE108022, FDR < 0.05). Each data point represents an individual sample. The horizontal line indicates the mean expression and asterisks denoting significance (*p < 0.05, **p < 0.01, and ***p < 0.001; n.s., not significant, Student’s t-test). (F) Immunohistochemical staining of PROX1 (left) and DHCR7 (right) in clinical RMS tumor samples obtained from the Helsinki Biobank. The tumors exhibit robust nuclear PROX1 expression and cytoplasmic DHCR7 expression compared to adjacent healthy muscle tissue (tumor border marked with a dashed line).

Article Snippet: Human RD cells (CCL-136) were acquired from the American Type Culture Collection (ATCC), while KLHEL1 cells were derived from a biopsy of a 27-year-old male diagnosed with high-grade alveolar rhabdomyosarcoma (ARMS).

Techniques: RNA Sequencing, Transduction, Extraction, Expressing, Muscles, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Targeting de novo cholesterol synthesis in rhabdomyosarcoma induces cell cycle arrest and triggers apoptosis through ER stress-mediated pathways

doi: 10.1101/2024.12.18.628900

Figure Lengend Snippet: Inhibition of cholesterol biosynthesis suppresses RMS cell growth and survival. (A) Quantitative PCR analysis of DHCR7 mRNA expression in RD cells following shSCR (control) and shDHCR7 transduction with three independent shRNA constructs. (B) Cell growth assessed by IncuCyte live cell imaging for stably expressing shSCR or shDHCR7 RD cells utilizing three different silencing constructs. (C, D) Colony formation assay and corresponding quantification in RD cells. (E) Quantification of cleaved caspase 3/7 activity in control and DHCR7-silenced RD cells using a fluorescent reporter and live imaging. (F) Growth curves of RD cells treated with different concentrations of DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. (G) Caspase 3/7 activity in dimethyl sulfoxide (DMSO) and DHCR7 inhibitor-treated RD cells. (H) qPCR analysis of DHCR7 mRNA expression in KLHEL1 cells following shSCR (control) and shDHCR7 transduction with three independent shRNA constructs. (I) Cell growth based on IncuCyte live cell imaging for stably expressing shSCR or shDHCR7 KLHEL1 cells. (J, K) Colony formation assay and corresponding quantification in KLHEL1 cells. (I) Caspase 3/7 activity in control and PROX1-silenced KLHEL1 cells utilizing a fluorescent reporter. (M) Growth curves of KLHEL1 cells treated with indicated concentrations of DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. (N) Caspase 3/7 activity in DMSO and DHCR7 inhibitor-treated KLHEL1 cells assessed using a fluorescent reporter. *p < 0.05, **p < 0.01, and ***p < 0.001. Data presented as mean ± standard error of the mean (SEM).

Article Snippet: Human RD cells (CCL-136) were acquired from the American Type Culture Collection (ATCC), while KLHEL1 cells were derived from a biopsy of a 27-year-old male diagnosed with high-grade alveolar rhabdomyosarcoma (ARMS).

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Expressing, Control, Transduction, shRNA, Construct, Live Cell Imaging, Stable Transfection, Colony Assay, Activity Assay, Imaging

Journal: bioRxiv

Article Title: Targeting de novo cholesterol synthesis in rhabdomyosarcoma induces cell cycle arrest and triggers apoptosis through ER stress-mediated pathways

doi: 10.1101/2024.12.18.628900

Figure Lengend Snippet: Inhibition of cholesterol synthesis impedes cell cycle progression. (A) Principal Component Analysis (PCA) illustrating the variance between the samples of DHCR7-silenced RD cells compared to control RD cells. (B) Sample distance analysis of RNA-seq samples (n = 4 + 4) depicting the relationship between DHCR7-silenced and control RD cells. (C) MA plot displaying Differentially Expressed Genes (DEGs) in red (FDR < 0.05, log2 fold change (FC) cutoff 0.58) in DHCR7-silenced RD cells compared to controls, encompassing 1,961 upregulated and 2,698 downregulated genes. (D-I) Gene Set Enrichment Analysis (GSEA) plots highlighting the most significantly affected functional categories among downregulated genes in DHCR7-silenced RD cells. The Normalized Enrichment Score (NES) and False Discovery Rate (FDR) values are provided for each category. (J - M) Propidium Iodide (PI) flow cytometry analysis of cell cycle distribution and corresponding statistical analysis in RD cells at first day (J and L) and second day (K and M) post-infection with DHCR7 shRNA. *P < 0.05. Data are presented as mean ± SEM.

Article Snippet: Human RD cells (CCL-136) were acquired from the American Type Culture Collection (ATCC), while KLHEL1 cells were derived from a biopsy of a 27-year-old male diagnosed with high-grade alveolar rhabdomyosarcoma (ARMS).

Techniques: Inhibition, Control, RNA Sequencing, Functional Assay, Flow Cytometry, Infection, shRNA

Journal: bioRxiv

Article Title: Targeting de novo cholesterol synthesis in rhabdomyosarcoma induces cell cycle arrest and triggers apoptosis through ER stress-mediated pathways

doi: 10.1101/2024.12.18.628900

Figure Lengend Snippet: Inhibition of cholesterol biosynthesis induces ER stress-mediated apoptosis. (A-E) Gene Set Enrichment Analysis (GSEA) plots illustrating the most significantly enriched functional categories among the upregulated genes in DHCR7-silenced RD cells. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are shown for each category. (F) Real-time qPCR analysis of genes’ expression levels involved in the ER stress-induced apoptotic pathway following DHCR7 knockdown in KLHEL cells. Data are presented as mean ± SEM. (G and H) Western blot analysis demonstrating the phosphorylation of eIF2α and the expression of proteins associated with the ER stress-induced apoptotic pathway following DHCR7 knockdown in RD cells (G) and KLHEL1 cells (H).*p < 0.05, **p < 0.01, and ***p < 0.001. Data are presented as mean ± SEM.

Article Snippet: Human RD cells (CCL-136) were acquired from the American Type Culture Collection (ATCC), while KLHEL1 cells were derived from a biopsy of a 27-year-old male diagnosed with high-grade alveolar rhabdomyosarcoma (ARMS).

Techniques: Inhibition, Functional Assay, Expressing, Knockdown, Western Blot, Phospho-proteomics

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 1. Biological activity of hLMP2 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#2 (T type) clone and flat revertants of the SKN-LMP2#122 (F type) clone (magnification 100). Changes in human uterine LMS cell line, SKN-transfectants, SKN-CEM9 (T type) clone, and SKN-LMP2wt (F type) clone xenograft volumes in mice (n = 8). Representative photographs of xenografts in mice (Left). Tumor growth of SKN-LMP2 was markedly reduced in comparison with that of the control transfectant SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9 (T type) clone and SKN-LMP2 (F type) clone (Right). (B) RT-PCR experiments revealed hLMP2, hLMP7, Calponin h1, SRF, cyclin B and b-actin mRNA expression in tumors. Precursor LMP2 or LMP7 (pre-LMP2, pre- LMP7) and mature LMP2 or LMP7 (LMP2, LMP7) are shown. (C) Western blotting revealed LMP2, LMP7, calponin h1, SRF, cyclin B, and b-actin in SKN-transfectant clones. (D) The luciferase reporter vectors containing the hCalponin h1 promoter with wild type SRF binding sites (Calponin-wt-Luc.), mutant SRF binding sites (Calponin-mut-Luc.), or empty luciferase reporter vector (Basic-Luc.) [23] were transiently co-transfected with pSV-b-galactosidase in SKN-transfectants, SKN-CEM9#2, SKN-LMP2#121, or SKN- LMP2#122 clones for the final 48 h, and then luciferase activities were measured. Values were normalized to those obtained with the co-transfected pSV-b-galactosidase expression vector. Each assay was performed at least three times and in triplicate. Luciferase reporter assays showed that LMP2 expression markedly induced calponin h1 promoter activation. Data are presented as the mean from three independent experiments (⁄S.D.). The experiments were performed four times with similar results. SKN transformantsa, CEM9 SKN-CEM9#2; LMP2, SKN-LMP2wt#121, SKN-LMP2wt#122. Detail is shown in SFig. 2, SFig. 3 and STable 3. RT-PCRb, total RNA samples were isolated from the individual xenografted-tumors, which were removed at 5 weeks after xenografting. W.B.c, W.B. are performed with the total cell lysates from SKN transformants.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, Comparison, Control, Transfection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Binding Assay, Mutagenesis, Plasmid Preparation, Activation Assay, Isolation

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 2. Biological activity of calponin h1 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#1Scr.shRNA (T type) clone, SKN-CEM9#2 calponin h1shRNA (T type) clone, SKN-CEM9#2 (T type) clone, SKN-LMP2#1Scr.shRNA (F type) clone, and SKN-LMP2#2Calponin h1shRNA (T type) clone of the SKN-LMP2 (F type) clone (magnification 60). The growth rates of the SKN-transfectant clones were measured as population doubling time (PDT). (B) Western blotting and RT-PCR experiments revealed calponin h1, precursor LMP2 (pre-LMP2), mature LMP2 (LMP2), and b-actin in SKN-transfectant clones. SKN transformantsa, CEM9#3 Scr.shRNA, CEM9#4 Calponin h1shRNA, LMP2#1 Scr.shRNA, LMP2#2 Calponin h1shRNA, Detail is shown in Table 1 and SFig. 5 and STable 3. (C) Changes in the human uterine LMS cell line, SKN-transfectant, SKN-CEM9#2 (T type) clone, SKN-LMP2wt#2/Calponin h1shRNA (T type) clone, and SKN-LMP2wt#1/ Scr.shRNA (F type) clone xenograft volumes in mice (n = 3). Representative photographs of xenografts in mice (Left). Tumor growth of the SKN-LMP2wt#2/Calponin h1shRNA (T type) clone is mildly increased in comparison with that of the SKN-LMP2wt#1/Scr.shRNA (F type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-transfectant clones (Right). RT-PCR experiments revealed hCalponin h1, hLMP2 and b-actin mRNA expression in tumors (Bottom). Experiments were performed three times with similar results. SKN-CEM9c, SKN- CEM9#2; LMP2wt+Calponin h1shRNAd, SKN-LMP2wt#2/ CalponinshRNA; LMP2wt/Scr.shRNAe, SKN-LMP2wt#1/Scr.shRNA. Details of SKN transfectants are shown in Table 1, SFig. 5 and STable 3. RT-PCRf, total RNA samples were isolated from the individual xenografted-tumors, which were removed from BALB/c nu/numice at 5 weeks after xenografting. Xenograftsg, BALB/c nu/nu mice were inoculated with SKN-CEM9#2, SKN-LMP2wt#2/CalponinshRNA or SKN-LMP2wt#1/Scr.shRNA.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, shRNA, Transfection, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Injection, Expressing, Isolation

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: Exploring downstream gene regulated by IMUP. a Heatmap and volcano plot analyzed using RNA-seq of BxPC-3 cells infected with shNC, IMUP-sh1, sh2, and sh3. b Gene ontology analysis of IMUP-correlated genes from TCGA database by LinkedOmics. c The intersection of negatively correlated genes (NCGs) from RNA-seq (fold-change > 2), NCGs from GeneChip, and NCGs from TCGA. d , e Pearson correlation of FHL1 and IMUP expression according to d the results of GeneChip and e TCGA data. f Kaplan–Meier analysis of OS by FHL1 expression data from TCGA

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: RNA Sequencing, Infection, Expressing

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: IMUP regulates cyclin A2/cyclin E1/CDK2 proteins via FHL1. a WB analysis of BxPC-3 and SW1990 cells infected with lentivirus carrying IMUP-sh1 or sh2. GAPDH and tubulin were used as a loading control. b WB analysis of BxPC-3 and SW1990 cells infected with lentivirus carrying IMUP-sh or co-infected with IMUP-sh and FHL1-siRNA. c Representative IHC staining of IMUP and FHL1 in 57 human PDAC samples. Case 1 and case 2 were two representative specimens analyzed as high and low expression of IMUP, respectively. (case 1, high IMUP and low FHL1; Case 2, low IMUP and high FHL1). Scale bars: 100 μm. Correlation between IMUP and FHL1 analyzed by Pearson correlation tests

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Infection, Control, Immunohistochemistry, Expressing

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: Knockdown of FHL1 rescues the phenotype inhibited by IMUP depletion. a , b , c , g BxPC-3 and SW1990 cells infected with shNC or IMUP-sh, or co-infected with IMUP-sh and FHL1-sh were used to analyze the a proliferation by cell Counting Kit-8, b tumorigenicity by mice xenograft ( n = 5/group; male), c colony formation capability by colony formation assay, and g cell cycle by flow cytometry. d Xenograft tumor volume, e colony formation, and h cell cycle were analyzed by two-way ANOVA test. f Tumor weight was analyzed by unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Knockdown, Infection, Cell Counting, Colony Assay, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: IMUP inhibits FHL1 transcription by NPM1-induced promoter methylation. a BxPC-3 cells were transfected with GFP-tagged IMUP. Exogenous co-immunoprecipitation was performed by GFP beads (left). Endogenous interaction was tested in BxPC-3 cells by anti-NPM1 (right). b WB analysis of proteins extracted from BxPC-3 infected with control shRNAs, IMUP sh1, or sh2. c Immunofluorescence double-staining of BxPC-3 cells showed the location of IMUP and NLM1. Cells were stained with anti-IMUP (green) and anti-NPM1 (red). The nuclei were stained with DAPI (blue). Scale bars: 50 μm. d BxPC-3 cells transfected with IMUP or control siRNAs were treated with 100 μg/mL CHX at the indicated time. Proteins were analyzed with anti-IMUP, anti-NPM1, and anti-tubulin. e The densitometric quantitation of NPM1 protein at the indicated time was normalized by tubulin. Statistical differences were analyzed by two-tailed Student’s t test (*** P < 0.0001). f Structure of the human FHL1 gene. Dotted lines indicate two exon fragments containing CpG islands and corresponding sequences. g The pyrosequencing maps of FHL1 promoter CpG islands. DNA was collected from BxPC-3 cells infected with control shRNAs, IMUP-shRNAs, or co-transfected with IMUP-shRNAs and NPM1 vectors. The methylation rates of FHL1 promoter CpG islands. Fragment 1 (left histogram) and fragment 2 (right histogram). Statistical differences were analyzed using two-way ANOVA test. *** P < 0.0001. h Translational factor SP1-binding motif and putative SP1-binding sequences of FHL1 promoter. i ChIP-qPCR revealed SP1 enrichment on the FHL1 promoter in BxPC-3 cells infected with either control shRNAs, or IMUP-shRNAs or co-transfected with IMUP-shRNAs and NPM1 vectors. The upstream of FHL1 promoter and actin promoter were used as negative controls. Statistical differences were analyzed by two-tailed Student’s t test (** P < 0.001, *** P < 0.0001)

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Methylation, Transfection, Immunoprecipitation, Infection, Control, Immunofluorescence, Double Staining, Staining, Quantitation Assay, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: FHL1 interacts with CHK1/CDC25A/14–3-3ξ and promotes the phosphorylation of CDC25A via CHK1. a WB analysis of input and anti-Flag IP derived from BxPC-3 cells transfected with Flag-FHL1 vectors. b Endogenous proteins from BxPC-3 were immunoprecipitated using anti-CDC25A, CDC25C, CHK1, 14–3-3ξ antibody, or rabbit IgG as a negative control. c WB analysis of BxPC-3 and SW1990 cells treated with control vectors, Flag-FHL1 vectors, or/and treated with 25 μmol CHK1 inhibitor (GDC-0575 analog). d BxPC-3 cells were treated with control vectors or Flag-FHL1 vectors. WB analysis of IP by anti-CHK1 or IgG

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Phospho-proteomics, Derivative Assay, Transfection, Immunoprecipitation, Negative Control, Control

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: FHL1 causes CDC25A to become sequestered in the cytoplasm via binding to 14–3-3ξ. a BxPC-3 and SW1990 cells were treated with control vectors, Flag-FHL1 vectors, or co-transfected with Flag-FHL1 vectors and 14–3-3ξ siRNAs. Nuclear-cytoplasmic isolated proteins were used for WB analysis. C, cytoplasm; N, nucleus. b BxPC-3 and SW1990 cells were treated with control siRNAs, IMUP-siRNAs, or co-transfected with IMUP siRNAs and 14–3-3ξ siRNAs. Nuclear-cytoplasmic isolated proteins were used for WB analysis. Right panels show the densitometric analysis of CDC25A distributed in the cytoplasm or nucleus in three independent experiments. Statistical differences (the ratio of nucleus to cytoplasm) were analyzed by unpaired Student’s t test. *** P < 0.0001. c BxPC-3 cells were transfected with control vectors or Flag-FHL1 vectors. WB analysis of IP by anti-14–3-3ξ or IgG. d Representative immunofluorescence analysis of xenograft mouse tumors infected with control shRNA and IMUP-sh1. Cells were stained with anti-CDC25A (red). Nuclei are stained with DAPI (blue). Scale bars: 100 μm. e Schematic of the mechanism underlying IMUP-regulated S phase progression through NPM1/FHL1-mediated cell-cycle kinase protein activation. IMUP enhances the stability of NPM1 by direct binding. NPM1 indirectly facilitates promoter CpG island methylation of FHL1 and inhibits the transcription of FHL1 . FHL1 promotes the phosphorylation of CDC25A by CHK1 and sequesters CDC25A in the cytoplasm by forming CDC25A/14–3-3ξ complexes

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Binding Assay, Control, Transfection, Isolation, Immunofluorescence, Infection, shRNA, Staining, Activation Assay, Methylation, Phospho-proteomics

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a , Top: schematic of hPSC directed differentiation to skeletal muscle in vitro and engraftment in vivo. Left: region of hPSC SMPCs 30 days post engraftment (1.44 mm 2 ) showing insets (rotated 90°) of chimeric and human-only myofibre phenotypes. Human-specific antibodies lamin AC (red) mark human nuclei, and dystrophin (green) mark human myofibres, 4′,6-diamidino-2-phenylindole (DAPI) (nuclei, blue). Scale bar, 200 μm. Right: Imaris was used to quantify myofibre cross-sectional area as one parameter of chimeric and human-only myofibres. Scale bar, 100 μm. Graph shows mean ± standard deviation of engrafted human and human–mouse myofibres over the length of mouse TA muscle ( N = 7). Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue). Scale bar, 50 μm. To quantify location of PAX7 cells, numbers were then quantified using 20× images (0.15 mm 2 ) were separated into four regions: (A) fields of view containing >50 human-only myofibres, (B) fields of view containing >10 chimeric muscle myofibre, (C) mouse myofibres, interstitial space and epimysium, and (D) dense numbers of human cells but few fused myofibres. Graph shows mean ± standard deviation of six to ten images from each region, N = 3 mice were quantified; statistics were performed using one-way ANOVA with post-hoc Tukey test, * P < 0.05.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques: In Vitro, In Vivo, Standard Deviation

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a . Human skeletal muscle tissues show foetal and adult PAX7 + SMPC/SCs in niches compared with hPSC SMPC engraftment. Immunofluorescent images show PAX7 (green), spectrin (red), laminin (grey) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Engrafted hPSC SMPCs are also stained with lamin AC (red) to identify human cells. N = 3 human tissue samples. Scale bar, 20 μm. b , Over time, PAX7 SMPCs associate within the basal lamina niches through association with human-only myofibres, which generate myofibre bundles integrated with spectrin cross bridges (arrows). Scale bar, 10 μm (left). Phenotypically, hPSC SMPC niches resemble foetal SMPC niches in vivo with formation of spectrin cross bridges within the myofibre bundles. Scale bar, 5 μm (right). c , Dynamics of SC niche occupancy by PAX7 + SCs and SMPCs 30 days post engraftment. Immunofluorescent images show representative location of PAX7 cells in chimeric SC niches, outside of chimeric SC niches, near human myofibres, or no niche interstitial space. Shown are PAX7 (green), lamin AC and spectrin (red), laminin (grey) and DAPI (blue). Scale bar, 5 μm. Graph of percentage mean ± standard error of the mean shows that adult SCs are better able to reside in niches of chimeric myofibres, while hPSC and foetal SMPCs are less efficient. N = 6 adult, N = 9 foetal, N = 14 hPSC SMPC engrafted tissues.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques: Staining, In Vivo

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a , Cartoon depicting hypotheses for SMPC association with human-only myofibres in the Pax7-ablation model (Pax7-ablation (DTX) mdx-NSG). Top: the occupied niche hypothesis predicts SMPCs cannot home to chimeric niches occupied by endogenous satellite cells, and the regenerative niche hypothesis predicts mouse SCs take up position in chimeric niches during regeneration. Bottom: engraftable Pax7 ablation mouse shows expected results for occupied niche and regenerative niche hypotheses. b , Cartoon of ablation mouse model generation and Pax7 cell numbers (white boxes) in Pax7-ablated and non-ablated control mice after 7 days of TMX treatment (resulting in Pax7 ablation or DTX), mean ± standard deviation, N = 5 mice, t -test * P = 0.0003. Tibialis anterior muscles of Pax7-ablated mice are atrophic; some individual myofibres of Pax7-ablated muscle undergo hypertrophy as measured by haematoxylin and eosin staining. c , Human foetal SMPCs increase PAX7 + numbers in Pax7-ablation mice. Representative images show co-staining of human nuclei (red), PAX7 cells (green) and 4′,6-diamidino-2-phenylindole (DAPI). White boxes indicate human PAX7 cells, and yellow boxes indicate mouse Pax7 SCs. Mean ± standard deviation of human PAX7 cells are quantified from Pax7-ablated and non-ablated mice, N = 5 per group, t -test, * P < 0.0004. d , Location of foetal PAX7 + cells are quantified 30 days post engraftment. Scale bar, 50 μm. Inset shows that human PAX7 foetal SMPCs are associated with small human-only myofibres relative to non-ablated controls, N = 5 per group, t -test, * P = 0.034.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques: Control, Standard Deviation, Muscles, Staining

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a , SMPC engraftment after 30 days show that human-only myofibres, but not chimeric myofibres express ACTC1 (green). N = 4 engrafted tissues. Scale bar, 100 μm. b , Human-only myofibres, but not chimeric myofibres, express regenerative signature as shown by ACTC1. SC niches form near small regenerating myofibres, which enables PAX7 cells to remain. Scale bar, 100 μm. Mouse TA express high levels of Myh4, which is not expressed by human, enabling further identification of human-only and chimeric myofibres. ACTC1 expression is reduced in chimeric Myh4 + spectrin + lamin AC + myofibres. c , Cartoon of repopulation experiments is shown. Top: representative images show fibre size in no injury or after re-injury (dystrophin (green), lamin AC/spectrin (red) or PAX7 (green). Insets show PAX7 location near other PAX7 foetal SMPCs. Fibre size is quantified using Imaris in non-injured or re-injured muscle. Bottom: graphs quantify mean ± standard deviation repopulation ability after re-injury, and dots represent individual images used for quantification; 10–15 images per sample, N = 3 adult, N = 4 foetal biological replicates per timepoint. PAX7 + SMPC/SCs and total human nuclei are quantified from the same image sets. t -test, * P < 0.05 between PAX7 + SMPC/SCs in chimeric or human-only myofibre niches. Data show that foetal SMPCs cannot repopulate after re-injury, but adult SCs can repopulate in mdx-NSG mice; however, in Pax7-ablated mdx-NSG mice in re-injury (D60 + RI) foetal SMPCs can repopulate both chimeric and human-only myofibres. t -test, * P < 0.006.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques: Expressing, Standard Deviation

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a , Regenerative ability is lost in Pax7-ablated mdx-NSG mice 8 days post injury (dpi) as shown by lack of eMyHC (left, green) and Pax7 (right, green). Inset shows mouse Pax7 SCs (asterisk) associate with small myofibres during regeneration. N = 3 mice. Scale bar, 50 μm. b , Schematic of experimental groups show timing in days (D) of TMX treatment resulting in Pax7 ablation (DTX) and injury (BaCl 2 ) of Pax7-ablated mdx-NSG mice used for snRNA-seq. UMAP of combined samples identifies 21 cell populations; abbreviations are defined in Supplementary Table ( N = 4). c , Left: graph shows percentage of all myonuclear (grey) and mononuclear (white) populations in response to injury or Pax7 ablation. Left centre: graphs show percentage of mononuclear cell types; boxes show enlarged view of satellite cells (SC.1) and myocytes (MC). Right: graphs show percentage of myonuclear populations; boxes show enlarged view of Car3 + Myh4 + myonuclei (IIB.2) and Actc1 + Myh4 + myonuclei (IIB.3). Bottom: UMAPs show spatial relationships and key genes of satellite cell, myocyte and myofibre populations during muscle regeneration. d , Gene Ontology of upregulated genes in Actc1 + myonuclei (IIB.3) is shown ( q < 0.05). UMAPs of key upregulated genes in IIB.3 myonuclei are shown. CellChat analysis shows laminin signalling pathway network between populations with specific interaction between IIB.3 myofibres and SC.1, SC.2 and MB.1.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques:

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a , Cartoon workflow of spatial transcriptomics on engrafted human/mouse tissue. Photocleavable RNA oligo probes are collected on ROIs for sequencing. Tissues used for spatial RNA-seq included foetal SMPCs engrafted in PAX7-ablated (or DTX) mdx-NSG mice for 60 or 120 days, and re-injury with CTX at 60 days. b , PCA plot showing clustering of engrafted human cells (PAX7 + , PAX7 − ) and myofibres (ACTC1 + , ACTC1 − ). Each dot represents a sequenced ROI ( N = 43). c , Top left: images taken on GeoMX of day 60 engrafted foetal SMPCs show ACTC1 + and ACTC1 − myofibres. GeoMx applied masks to ROIs used for sequencing. Right: volcano plot of ACTC1 + and ACTC1 − myofibres. Red dots represent differential expressed genes, and selected key genes are shown. For all volcano plots differential gene expression (DGE) between ROIs was determined using GeoMx linear mixed model (LMM) statistical tests with Benjamini–Hochberg (BH) correction P < 0.05. Bar graphs show Gene Ontology of selected pathways upregulated by ACTC1 + and ACTC1 − myofibres. Bottom left: PAX7 in the re-injury group and regions used for selecting single PAX7 cells for sequencing. d , Volcano plot of human foetal PAX7 + SMPCs after re-injury or no-injury 120 days after engraftment. Red dots represent differentially expressed genes, and selected key genes are shown. Graphs show Gene Ontology of foetal PAX7 + SMPCs undergoing repopulation after re-injury of selected pathways related to the SC niche comparisons between two groups. For comparing different cell types or treatments to generate volcano plot data, LMM statistical tests with BH correction were performed. Neg. reg., negative regulation. Right: CellChat analysis show predicted sender (bottom) and receiver (top) ligand and receptor interactions between foetal PAX7 + SMPCs and ACTC1 + and ACTC1 − myofibres from spatial RNA-seq of engrafted tissues.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques: Sequencing, RNA Sequencing, Gene Expression

Journal: Nature Cell Biology

Article Title: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

doi: 10.1038/s41556-023-01271-0

Figure Lengend Snippet: a , Cartoon showing iCaspase9 vector (iCasp9) insertion immediately in 3′ of ACTC1 locus of hPSCs. AP1903 causes iCasp9 activation and gene-specific cell death. PCR products show insertion of iCasp9 across two hPSC lines (H1 and H9). b , Top: Pax7-DTX (ablated) Mdx-D2-NSG mice were treated with TMX, and hPSC SMPCs transplanted into Pax7-ablated tissues. Mice were treated six times every two weeks with AP1903 over 90 days, N = 5 cross sections. Left: images taken on the GeoMx of 90-day engrafted hPSC SMPCs show regions of transplantation with or without AP1903 treatment. Zoom shows morphology markers and masked ROI overlays. Single PAX7 cells and myofibres were collected for spatial RNA-seq. c , Graph show mean ± standard deviation of PAX7 counts from cross-sectional area and a decrease in AP1903-treated mice; t -test,*** P = 0.0005, N = 5 cross sections. ACTC1 expression counts were obtained from Q3 normalized outputs from ROIs on GeoMX; ** P < 0.003, N = 13 untreated, N = 8 AP1903. d , Top: volcano plot of all human ROIs in control and AP1903-treated group highlight increased caspase activity in AP1903-treated mice. Bottom: volcano plot in control mice shows genes upregulated hPSC PAX7 + SMPCs compared with hPSC myofibres. Red dots represent differentially expressed genes, and selected key genes are shown. For comparing different cell types or treatments to generate volcano plot data, linear mixed model (LMM) statistical tests with Benjamini–Hochberg (BH) correction were performed. e , CellChat analysis show predicted sender (bottom) and receiver (top) ligand and receptor interactions between hPSC PAX7 SMPCs and hPSC myofibres from spatial RNA-seq of engrafted tissues.

Article Snippet: Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin. b , 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).

Techniques: Plasmid Preparation, Activation Assay, Transplantation Assay, RNA Sequencing, Standard Deviation, Expressing, Control, Activity 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) 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