Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Immunofluoresence (green) detection of δ-, α-, β-, and γ-sarcoclycan (SGC), β-dystroglycan (β-DG), utrophin (Utro.), dystrophin (Dystro.) and β1D-integrin in littermates of three month-old WT, Thbs4-Tg, Sgcd -/- and Sgcd -/- Thbs4-Tg quadriceps. Representative images of 4 mice per genotype are shown. Scale bar = 25 μm. ( B ) Representative Western blots of sarcolemmal protein extracts (upper) or total cytoplasmic protein extracts (lower) from the quadriceps of the indicated groups of mice for the indicated proteins (n = 4–5 biological replicates). Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5- integrin. The red boxes show increased protein levels. Also see for replicates. ( C ) Representative immunoblotting for structural components of the DGC- and integrin-associated protein complexes in sarcolemmal preparations from Thbs4 -/- and WT quadriceps at four months of age (n = 4 biological replicates). The burgundy-boxed areas show reduced protein levels. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls for sarcolemmal protein extracts; Gapdh was used as loading control for total cell protein extracts. DOI: http://dx.doi.org/10.7554/eLife.17589.018

Article Snippet: Primary antibodies included: δ-sarcoglycan (Abcam, ab92896, 1:100), α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan (NovaCastra, Buffalo Grove, IL, NCL-a-sarc, NCL-b-sarc and NCL-g-sarc, all 1:250), β-dystroglycan (Development Studies Hybridoma Bank, Iowa City IA, MANDAG2; 1:50), utrophin (Santa Cruz Biotechnology, MANCHO7, sc-81557; 1:50), dystrophin (Abcam, ab15277, 1:200) and β1D-integrin (Millipore, Billerica, MA, MAB1900, 1:250).

Techniques: Western Blot, Staining, Control

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A , B ) Representative Western blots of sarcolemmal protein extracts from the quadriceps of the indicated groups of mice at three months of age (n = 4–5 biological replicates). Sgcd -/- Tg and mdx Tg indicate Sgcd -/- and mdx with skeletal muscle specific Thbs4 overexpression, respectively. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls. Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-itg (integrin). ( C ) Immunoblots for β1D- and α7-Integrin (Itg), β-dystroglycan (DG) and Thbs4 (Flag) from neonatal rat ventricular myocyte extracts immunoprecipitated with a Flag antibody (Thbs4). Adβgal was used as a control infection (n = 3 biological replicates). An adenovirus expressing a Flag-tagged Thbs4 protein was used to achieve high level of this protein to identify the interaction. ( D , E ) Representative Western blots for Thbs4, α5- and β1D-integrin (itg) from intracellular vesicular isolates from WT and Thbs4 Tg quadriceps ( D ) or WT and Sgcd -/- quadriceps ( E ) that were immunoprecipitated with an antibody raised against the cytoplasmic domain of β1D-integrin (n = 3 biological replicates), showing that Thbs4 and α5-integrin localize to β1D-integrin-positive intracellular vesicles. α-tubulin and Gapdh are presented as loading control. DOI: http://dx.doi.org/10.7554/eLife.17589.019

Article Snippet: Primary antibodies included: δ-sarcoglycan (Abcam, ab92896, 1:100), α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan (NovaCastra, Buffalo Grove, IL, NCL-a-sarc, NCL-b-sarc and NCL-g-sarc, all 1:250), β-dystroglycan (Development Studies Hybridoma Bank, Iowa City IA, MANDAG2; 1:50), utrophin (Santa Cruz Biotechnology, MANCHO7, sc-81557; 1:50), dystrophin (Abcam, ab15277, 1:200) and β1D-integrin (Millipore, Billerica, MA, MAB1900, 1:250).

Techniques: Western Blot, Over Expression, Staining, Immunoprecipitation, Control, Infection, Expressing

Journal: eLife

Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

doi: 10.7554/eLife.17589

Figure Lengend Snippet: ( A ) Schematic diagram of the transgene (Tg) used to overexpress ATF6α in skeletal muscle. ( B ) Western blot analysis for Thbs4, ATF6α, BiP, PDI and calreticulin (Calret.) expression in quadriceps (Quad) from WT, Thbs4 Tg and ATF6α Tg mice at six weeks of age. Gapdh is a processing and loading control (n = 6 biological replicates). ( C ) Masson’s trichrome-stained histological sections from Quad of WT and ATF6α-Tg littermates at six weeks of age. Representative images of 5 mice per genotype are shown. Scale bar = 100 μm. ( D ) Transmission electron micrographs in Quad from WT and ATF6α-Tg mice at six weeks of age. The white arrows show dramatic expansion of ER and associated vesicles throughout the cell and especially in the sub-sarcolemmal region. Representative images of 2 mice per genotype are shown. Scale bar = 2 μm. ( E ) Masson’s trichrome stained histological sections of Quad from Sgcd -/- and Sgcd -/- ATF6α Tg mice at six weeks of age. Representative images of 5 mice per genotype are shown. Scale bar = 100 μm. ( F ) Quantitation of serum CK levels (units/liter) in the indicated genotypes of mice shown in the legend below the graph at six weeks of age. n = 10 mice for WT and n = 8 mice for the remaining genotypes. *p<0.05 versus WT by one-way ANOVA with post hoc Tukey’s test. ( G , H ) Histological analysis of the Quad showing percentage of myofibers with centrally located nuclei (n = 5 mice for WT and ATF6α Tg, and n = 6 mice for Sgcd -/- and Sgcd -/- ATF6α Tg) and interstitial fibrosis (n = 5 mice per genotype) at six weeks of age in WT, ATF6α Tg, Sgcd -/- and Sgcd -/- ATF6α Tg mice. *p<0.05 versus WT by one-way ANOVA with post hoc Tukey’s test. ( I ) Time to fatigue in seconds with forced downhill treadmill running in the indicated genotypes of mice shown in the legend. n = 6 mice per genotype. *p<0.05 versus WT by one-way ANOVA with post hoc Tukey’s test. ( J ) Representative immunofluorescent images of EBD (red) uptake in myofibers in the Quad of six week-old mice of the indicated genotypes. Membranes of myofibers are shown in green. Scale bars = 75 μm. Percent EBD-positive myofibers is indicated. Six mice per genotype were analyzed for EDB uptake. ( K ) Western for structural components of the DGC and integrin-associated protein complexes in sarcolemmal protein preparations from Quad of WT, ATF6α Tg, Sgcd -/- and Sgcd -/- ATF6α Tg littermates at six weeks of age. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls (n = 3 biological replicates). Abbreviations: Utro, utrophin; Dystro, dystrophin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-Itg (integrin). All data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.17589.020

Article Snippet: Primary antibodies included: δ-sarcoglycan (Abcam, ab92896, 1:100), α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan (NovaCastra, Buffalo Grove, IL, NCL-a-sarc, NCL-b-sarc and NCL-g-sarc, all 1:250), β-dystroglycan (Development Studies Hybridoma Bank, Iowa City IA, MANDAG2; 1:50), utrophin (Santa Cruz Biotechnology, MANCHO7, sc-81557; 1:50), dystrophin (Abcam, ab15277, 1:200) and β1D-integrin (Millipore, Billerica, MA, MAB1900, 1:250).

Techniques: Western Blot, Expressing, Control, Staining, Transmission Assay, Quantitation Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Sequence of primers used in the PCR assays

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Sequencing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: 2OHOA inhibits Notch signaling pathway. a Representative immunoblots of Notch2/3 FL (full length), Notch2/3 TM (transmembrane domain) and NICD2/3 (Notch intracellular domain) proteins after exposure of U-87 MG cells to 2OHOA (200 µM) for 6, 12, 24 and 48 h (four independent experiments with two replicates each). b The Hes1 protein levels after exposure to 2OHOA (200 µM) for 48 h as an indicator of its pharmacological effect in U-87 MG cells. c The mRNA expression of JAGGED1 , NOTCH1 , NOTCH2 , NOTCH3 , HES1 and CD3 genes in U-87 MG treated for 48 h with 2OHOA (200 µM) or the vehicle alone. The values are presented as the mean ± SEM of at least four independent experiments analyzed in triplicate. d JAGGED1 mRNA levels in U-87 MG cells exposed to 2OHOA (200 µM) or the vehicle alone for 72 h. The results are from three independent experiments with three replicates each and they are expressed as the % of the control ± SEM. e Cell localization of Notch3 (left panels) and Hes1 (right panels) in U-87 MG control cells (row 1, 2) or those exposed to 2OHOA (400 µM) for 48 h (row 3, 4). Images were taken at 40× magnification. Scale bar: 20 μm (row 1, 3), 5 μm (row 2,4). f Nuclear fluorescence intensity of Notch3 (upper graph) or Hes1 (lower graph) in two independent experiments. The statistical analysis was performed with a t -test with Welch’s correction relative to the untreated cells: *** p < 0.001

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Western Blot, Expressing, Control, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: 2OHOA prevents Notch2 processing and trafficking. a Representative immunoblot of the U-87 MG subcellular fractionation from six independent experiments, with two replicates each. The distribution of the Notch protein: Notch2/3 FL (full length), Notch2 TM (transmembrane domain) and NICD3 (Notch intracellular domain) in cells maintained for 48 h in the presence or absence of 2OHOA (400 µM) or DAPT (10 µM, 24 h), a positive control for the loss of NICD, is shown in the different cellular compartments: N, nucleus; CT, cytosol; and MB, total membranes. The efficiency of fractionation was assessed by analyzing reference proteins from the different compartments: Laminin B1 for the nucleus, tubulin for the cytosol, and ATPase α (Na + /K + ) for the membranes. b U-87 MG immunofluorescence staining and co-localization analysis ( c ) of Notch2 (green channel) with Giantin (Golgi marker, red channel) in response to 400 µM for 48 h of vehicle (row 1, 2) or 2OHOA (row 3, 4). Images were taken at 63× magnification. Scale bar: 20 μm (row 1, 3), 5 μm (row 2, 4). Pearson correlation coefficient indicate the probability that the pixels of both channels coincide. Manders’ coefficients of M1 (overlap of Notch2 with Giantin) and M2 (overlap of Giantin with Notch2 signal). The values are given as the mean ± SEM and significant differences between the treatment and vehicle were analyzed with a Welch’s t -test: *** p < 0.001

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Western Blot, Fractionation, Positive Control, Immunofluorescence, Staining, Marker

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Graphical illustration describing the 2OHOA mechanism of action to inhibit Notch2 and Notch3 signaling. Model for activated Notch2/3 signaling (left panel). Proposal model for 2OHOA mechanism of action (right panel) where its interaction with the furin enzyme prevents Notch2 processing in Golgi. Instead, Notch3 is transcriptionally repressed probably by indirect 2OHOA mechanism (indicated as dashed line). In addition, HES1 overexpression partially inhibits the 2OHOA effect on viability (narrower dashed line). Continuous lines indicate activation while transparent continuous lines represent lower activation of the pathway

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Over Expression, Activation Assay