Journal: Nucleic Acids Research

Article Title: Deletion of Endonuclease V suppresses chemically induced hepatocellular carcinoma

doi: 10.1093/nar/gkaa115

Figure Lengend Snippet: Role of EndoV in apoptosis. ( A ) Response to sorafenib treatment in human Flp-In T-REx 293 cells overexpressing human Flag-EndoV (hEV; white bar) or human Flag-EndoV D52A (D52A; grey bar). Flp-In T-REx 293 cells are included as a control (Ctr; black bar). Apoptosis was assessed by fluorescently measuring the CASP3/7 activity for 24 h after sorafenib addition (4 μM). CASP3/7 positive cells were normalized to the total number of cells per well and related to the average of untreated sample for each cell line. Graphs are shown as means ± SEM ( n = 4). ( B ) Western blot of Flp-In T-REx cells (Ctr) overexpressing either human Flag-EndoV (hEV) or Flag-EndoV D52A (D52A) probed with a hEndoV antibody (upper panel). Molecular weight marker (M) with sizes (in kDa) is shown to the left. Probing with α-Tubulin (α-Tub) was used as a loading control (lower panel).

Article Snippet: Cells were subsequently monitored in real-time by fluorescently measuring the CASP3/7 activity for 24 h using IncuCyte ® S3 and IncuCyte caspase-3/7 Green Apoptosis Assay according to the manufacturer's protocol (Essen Bioscience).

Techniques: Activity Assay, Western Blot, Molecular Weight, Marker

Journal: Cell reports

Article Title: MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

doi: 10.1016/j.celrep.2023.113120

Figure Lengend Snippet: (A) Graphical representation of DUX4 nuclear interactors identified by proteomics. Proteins identified in all the STREP-HA affinity purifications with a spectral count average of DUX4/EV control ratio >4 are displayed. DUX4 is highlighted in yellow, and the interactors are displayed in gray. The thickness of the edges is proportional to the spectral count average of DUX4/EV ratio. (B) Quantitative real-time quantitative PCR showing the efficiency of knockdown for the indicated DUX4 interactors in HEK293 cells. Values are expressed relative to cells transfected with control small interfering RNAs (siRNAs; siNTs) (unpaired Student’s t test, *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001, n = 3). (C) Caspase-3/7 activity assays performed upon knockdown of the indicated DUX4 interactors in HEK293 cells not expressing DUX4 (paired Student’s t test, n = 4). (D) Caspase-3/7 activity assays performed in HEK293 cells collected 48 h after transfection with empty vector (EV), DUX4 , or DUX4 in combination with siRNAs specific for the indicated targets (paired Student’s t test, *p < 0.05, n = 5). (E) Caspase-3/7 activity assays performed in HEK293 cells collected 48 h after transfection with EV, DUX4 , or DUX4 in combination with expression vectors for the indicated factors (paired Student’s t test, **p < 0.01, ***p < 0.001, n = 4). See also and and .

Article Snippet: Briefly, 24 h post-transduction, differentiation medium was replaced with fresh medium containing 1ml/ml of IncuCyte CASP3/7 assay GREEN reagent (Essen BioScience) and the green fluorescence signal was acquired by the IncuCyte S3 Imager system as follows: 36 scans/well every 3 h for 72 h. The signal was analyzed using the IncuCyte software.

Techniques: Control, Real-time Polymerase Chain Reaction, Knockdown, Transfection, Activity Assay, Expressing, Plasmid Preparation

Journal: Cell reports

Article Title: MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

doi: 10.1016/j.celrep.2023.113120

Figure Lengend Snippet: (A) Live-cell, real-time, caspase-3/7 apoptotic assays on primary FSHD muscle cells transduced with CTRL or MATR3 lentiviruses. Live-cell imaging was performed by IncuCyte S3 Imager system and quantified using the IncuCyte software. Data are reported as a percentage (%) of green-fluorescent apoptotic cells normalized to time 0 (two-way ANOVA, **p < 0.01, ***p ≤ 0.001, n = 3). (B) Quantification of the area under the curve in (A) (unpaired Student’s t test, **p < 0.01). (C) Live-cell, real-time, caspase-3/7 apoptotic assays on primary FSHD muscle cells transfected with control (siNT) or MATR3 (si MATR3 ) siRNAs. Live-cell imaging was performed by IncuCyte S3 Imager system and quantified using the IncuCyte software. Data are reported as a percentage (%) of green-fluorescent apoptotic cells normalized to time 0 (two-way ANOVA, **p < 0.01, ****p ≤ 0.0001, n = 3). (D) Quantification of the area under the curve in (C) (unpaired Student’s t test, ****p ≤ 0.0001). (E) Representative images of myosin heavy-chain (MyHC; green) and nuclei (Hoechst 33342, blue) staining performed on differentiated primary FSHD muscle cells transduced with CTRL or MATR3 lentiviruses. Scale bar: 100 μm. (F) Quantification of differentiation index, fusion index, and nuclei distribution in primary FSHD muscle cells treated as in (E) (unpaired Student’s t test, *p < 0.05, **p < 0.01, ***p ≤ 0.001, n = 3). (G) Representative images of MyHC (green) and nuclei (Hoechst 33342, blue) staining performed on differentiated primary FSHD muscle cells transfected with control (siNT) or MATR3 (si MATR3 ) siRNAs. Scale bar: 100 μm. (H) Quantification of differentiation index, fusion index, and nuclei distribution in primary FSHD muscle cells treated as in (G) (unpaired Student’s t test, **p < 0.01, n = 3). See also and .

Article Snippet: Briefly, 24 h post-transduction, differentiation medium was replaced with fresh medium containing 1ml/ml of IncuCyte CASP3/7 assay GREEN reagent (Essen BioScience) and the green fluorescence signal was acquired by the IncuCyte S3 Imager system as follows: 36 scans/well every 3 h for 72 h. The signal was analyzed using the IncuCyte software.

Techniques: Transduction, Live Cell Imaging, Software, Transfection, Control, Staining

Journal: Cell reports

Article Title: MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

doi: 10.1016/j.celrep.2023.113120

Figure Lengend Snippet: (A) Schematic representation of MATR3 full length (MATR3-fl) and MATR3 deletion mutants MATR3 2–798 , MATR3 2–322 , MATR3 2–288 , and MATR3 289–847 . The N-terminal yellow box indicates the FLAG tag. (B) Immunoblotting on total protein extracts from HEK293 cells transfected with EV, DUX4 , or DUX4 in combination with the indicated MATR3 constructs, incubated with anti-MATR3 (recognizing endogenous as well as transfected MATR3 ), anti-HA (recognizing transfected DUX4 ), and anti-tubulin (as loading control). (C) Caspase-3/7 assay in HEK293 cells transfected with EV, DUX4 , and DUX4 in combination with the indicated MATR3 constructs (unpaired Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 10). (D) FLAG-immunoprecipitation (IP) on total protein extracts from HEK293 cells transfected with EV, MATR3-fl , or the indicated MATR3 constructs in combination with DUX4 , incubated with anti-DUX4 and anti-FLAG (recognizing transfected MATR3 constructs). (E) Pull-down assay with recombinant, purified His-DUX4 dbd plus purified GST-MATR3 2–288 or GST (as negative control), analyzed by immunoblotting with antibodies against GST or His tag. Asterisks (*) indicate the position of two degradation products of GST-MATR3 2–288 . See also and .

Article Snippet: Briefly, 24 h post-transduction, differentiation medium was replaced with fresh medium containing 1ml/ml of IncuCyte CASP3/7 assay GREEN reagent (Essen BioScience) and the green fluorescence signal was acquired by the IncuCyte S3 Imager system as follows: 36 scans/well every 3 h for 72 h. The signal was analyzed using the IncuCyte software.

Techniques: FLAG-tag, Western Blot, Transfection, Construct, Incubation, Control, Immunoprecipitation, Pull Down Assay, Recombinant, Purification, Negative Control

Journal: Cell reports

Article Title: MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

doi: 10.1016/j.celrep.2023.113120

Figure Lengend Snippet: (A) Quantitative real-time quantitative PCR for the indicated genes performed on RNA from differentiated primary FSHD muscle cells transduced with a CTRL or with MATR3 2–288 lentiviruses. Data are represented as relative to CTRL (unpaired Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3). (B) Volcano plot showing the significantly downregulated genes by MATR3 2–288 overexpression among the lists of DUX4 upregulated targets from Jagannathan et al., filtered for absolute values of |log2 fold change (FC)| >1. (C) Volcano plot showing the significantly upregulated genes by MATR3 2–288 overexpression among the lists of DUX4 downregulated targets from Jagannathan et al., filtered for absolute values of |log2 FC| >1. (D) Live-cell, real-time, caspase-3/7 apoptotic assays on primary FSHD muscle cells transduced with CTRL or MATR3 2–288 lentiviruses. Live-cell imaging was performed by IncuCyte S3 Imager system and quantified using the IncuCyte software. Data are reported as a percentage (%) of green-fluorescent apoptotic cells normalized to time 0 (two-way ANOVA, *p < 0.05, ****p ≤ 0.0001, n = 3). (E) Quantification of the area under the curve in (D) (unpaired Student’s t test, ***p ≤ 0.001). (F) Representative images of MyHC (green) and nuclei (Hoechst 33342, blue) staining performed on differentiated primary FSHD muscle cells transduced with CTRL or MATR3 2–288 lentiviruses. Scale bar: 100 μm. (G) Quantification of differentiation index, fusion index, and nuclei distribution in primary FSHD muscle cells treated as in (F) (unpaired Student’s t test, *p < 0.05, **p < 0.01, n = 3). See also and and .

Article Snippet: Briefly, 24 h post-transduction, differentiation medium was replaced with fresh medium containing 1ml/ml of IncuCyte CASP3/7 assay GREEN reagent (Essen BioScience) and the green fluorescence signal was acquired by the IncuCyte S3 Imager system as follows: 36 scans/well every 3 h for 72 h. The signal was analyzed using the IncuCyte software.

Techniques: Real-time Polymerase Chain Reaction, Transduction, Over Expression, Live Cell Imaging, Software, Staining

Journal: Cell reports

Article Title: MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

doi: 10.1016/j.celrep.2023.113120

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, 24 h post-transduction, differentiation medium was replaced with fresh medium containing 1ml/ml of IncuCyte CASP3/7 assay GREEN reagent (Essen BioScience) and the green fluorescence signal was acquired by the IncuCyte S3 Imager system as follows: 36 scans/well every 3 h for 72 h. The signal was analyzed using the IncuCyte software.

Techniques: Virus, Recombinant, Electron Microscopy, Luminescence Assay, In Situ, Mutagenesis, Transfection, SYBR Green Assay, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Dysregulation of RNA Splicing in Tauopathies

doi: 10.1016/j.celrep.2019.11.093

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Casp 3/7 Glo caspase activation kit , Promega , G8093.

Techniques: Recombinant, Activation Assay, XTT Assay, SYBR Green Assay, Reverse Transcription, Sample Prep, Software