Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

doi: 10.1128/JVI.76.8.4008-4021.2002

Figure Lengend Snippet: Stability of sfl genomes during continuous cell culture. (A) Six different cell lines were continuously cultured in the presence of G418. At the indicated time points (days [d]), cells were lysed, total RNA was prepared, and 4 μg was analyzed by Northern blotting. For comparison, given amounts of RNA containing subgenomic replicon transcribed in vitro were analyzed in parallel. To allow detection of sequence variants and of smaller HCV RNA pieces, the entire blot was hybridized under low-stringency conditions. (B) Genetic drift of the sfl genome in cell line 21-5. Cells were continuously cultured, and after 57 passages (corresponding to ∼6 months), total RNA was prepared. The coding region of the HCV polyprotein was amplified by long-distance RT-PCR in two overlapping fragments shown as bars below the ORF. For each fragment, two independent clones were sequenced. The boundary of the sequenced clones is indicated by a dashed vertical line. Amino acid substitutions are drawn as vertical lines at their respective positions within the HCV polyprotein. The hypervariable region 1 (HVR1) present at the N terminus of E2 is depicted as a shaded area. Six mutations were conserved between both sequenced clones, and they are indicated (stars).

Article Snippet: For cell lines carrying HCV replicons, various concentrations of G418 (Geneticin; Life Technologies GmbH) were added to the culture medium (100 μg/ml for cell line 20-1; 250 μg/ml for cell lines 21-5 and 21-7; 500 μg/ml for cell lines 22-1, 22-2, 22-6, and 22-16).

Techniques: Cell Culture, Northern Blot, In Vitro, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Clone Assay

Journal: The Science of the Total Environment

Article Title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment

doi: 10.1016/j.scitotenv.2021.145903

Figure Lengend Snippet: Data obtained from the chosen articles.

Article Snippet: , 43. Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care , USA , No , Surface and air samples from COVID-19 patient rooms , Air sampling: Sartorius Airport MD8 air sampler operating at 50 Lpm for 15 min. Surface samples: sterile swabs , Viral RNA Extractions: using a Qiagen DSP Virus Spin Kit. RT-qPCR: using Invitrogen Superscript III Platinum One-Step Quantitative RT-qPCR System. Primers and probe used target the E gene of SARS-CoV-2. , • We detected viral contamination among all samples. , ( ) .

Techniques: Sampling, Lysis, RNA Extraction, Environmental Monitoring, Virus, Multiplex Assay, Northern Blot, Marker, RNA Detection, Isolation, Membrane, Control, Environmental Sampling, Amplification, Transmission Assay, Aerosol, Diagnostic Assay, Infection, Sterility, Real-time Polymerase Chain Reaction, Nested PCR, Reverse Transcription, Extraction, Purification, Digital PCR, Preserving, Quantitative RT-PCR, cDNA Synthesis, Magnetic Beads, Incubation, Modification, One Step RT-PCR, Cell Culture, Sequencing

Journal: Viruses

Article Title: Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2

doi: 10.3390/v12060582

Figure Lengend Snippet: Summary table of available protocols posted to the WHO’s website.

Article Snippet: National Institute of Infectious Diseases, Japan , N gene , NR , , , Average Cq value of specimen was 36.7 and 35.0 for the positive control (500 copies of RNA transcript) , , , NR , 1 μL of 20 xprimer and probe mix in a 20 μL reaction with 5 μL of RNA. F primer at 500 nM, R primer at 700 nM, probe at 200 nM. , RNA extracted using QIAamp viral RNA mini kit (Qiagen). Reverse transcription via Super Script IV Reverse Transcriptase (Thermo). RT-PCR via QuantiTect Probe RT-PCR Kit (Qiagen).

Techniques: Amplification, Concentration Assay, RNA Extraction, Virus, Sequencing, Extraction, Diagnostic Assay, Positive Control, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, One Step RT-PCR, Cell Culture

Journal: Cell reports

Article Title: Oncogenic Vav1-Myo1f induces therapeutically targetable macrophage-rich tumor microenvironment in peripheral T cell lymphoma

doi: 10.1016/j.celrep.2022.110695

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RNeasy mini kit , Qiagen , 74106.

Techniques: Control, Recombinant, Adjuvant, Extraction, Liposomes, Plasmid Preparation, Cell Isolation, Cell Viability Assay, Reverse Transcription, Sequencing, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Differentiation induced by treatment with the Gsk3 inhibitor in hPSCs is β-catenin dependent. (A) H9-7TGP cells were treated with 12 μM CH in mTeSR1 for 4 d. Immunofluorescent staining for Oct4, Isl1, and Nkx2.5 was compared with GFP expression. (Scale bars, 50 μm.) (B and C) 19-9-11 shcat-2 and scramble cells were cultured on Matrigel with mTeSR1 medium containing 12 μM CH for 4 d. (B) RT-PCR analysis of pluripotent, mesendoderm, early mesoderm, and early cardiac gene expression was performed. (C) Oct4 expression on day 4 was analyzed by flow cytometry. Each colored line represents an independent replicate. n = 3. (D) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing CH. After 2 d, the expression of T in the scramble relative to its expression in the shcat-2 line was quantified by quantitative PCR. (E) Flow cytometry analysis of brachyury expression in 19-9-11 shcat-2 and scramble cells exposed to CH for 4 d. Error bars represent SEM of three independent replicates. (F) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing 12 μM CH. After 4 d, cells were immunostained for Nanog and Isl1. (Scale bar, 50 μm.)

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Staining, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Temporal regulation of Wnt/β-catenin signaling promotes cardiac differentiation induced by serum or growth factors. (A) H9 cells on MEFs were treated with CH in hESC medium for 3 d before forming EBs. EBs were cultured in suspension using serum containing medium for 4 d before being transferred to 0.1% (wt/vol) gelatin-coated plates. The percentage of contracting EBs was determined visually. (B) H9 cells were cultured on Matrigel and treated with DMSO, 1 μM CH, or 1 μM BIO for 3 d before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1 in RPMI/B27-insulin medium using monolayer-directed differentiation. At day 15, the percentage of cTnT+ cells in culture was assessed by flow cytometry. #P < 0.005, CH versus DMSO or BIO versus DMSO; Student’s t test. (C) Schematic of the inducible shRNA construct for β-catenin knockdown and shRNA sequences targeting β-catenin. PH1TetO represents the human H1 promoter with Tet operator sequences. Red and green sequences are forward and reverse shRNA sequences of β-catenin, respectively; the loop sequence is shown in blue. (D) Representative phase-contrast and mCherry epifluorescence images of 19-9-11 cells transduced with lentiviral vectors containing the constructs described in C and selected by puromycin treatment. (E) 19-9-11 ishcat-1 and ishcat-2 cells were cultured in mTeSR1 containing 2 μg/mL dox. After 3 d, mRNA was collected, and β-catenin expression was evaluated by quantitative PCR. Error bars represent SEM of three samples. #P < 0.005, ishcat-1 versus iscramble or ishcat-2 versus iscramble; Student’s t test. (F) 19-9-11 ishcat-1 cells were cultured in mTeSR1 medium and were treated with BIO before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1, with 2 μg/mL dox added at the indicated times. Cells were analyzed for cTnT expression by flow cytometry 15 d after initiation of differentiation. Error bars represent SEM. of three independent experiments. #P < 0.005, for each time point versus no dox; Student’s t test.

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Cell Culture, Flow Cytometry, shRNA, Construct, Sequencing, Transduction, Expressing, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Modulating regulatory elements of Wnt signaling is sufficient for efficient and reproducible generation of human cardiomyocytes in the absence of growth factors. (A) Schematic of protocol for defined, growth factor-free differentiation of hPSCs expressing dox-inducible β-catenin shRNA to cardiomyocytes via treatment with small molecules. (B and C) 19-9-11 ishcat-1 cells were cultured as indicated in A with dox added 36 h after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry (B) or immunofluorescence (C). In B, the green histogram represents cTnT expression, and the red histogram is an isotype control. (Scale bar in C, 50 μm.) (D) 19-9-11 ishcat-2 cells were cultured as indicated in A, with dox added at different time points after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry. Error bars represent SEM of three independent experiments. *P < 0.05 and #P < 0.005, each time point versus no dox; Student’s t test. (E and F) 19-9-11 ishcat-1 cells were differentiated as described in A, with dox added 36 h after treatment with 12 μM CH. (E) At different time points, mRNA was collected, and RT-PCR analysis of pluripotent, mesendoderm, mesoderm, and cardiac gene expression was performed. (F) Day 7 cells were analyzed for Isl1 and Nkx2.5 expression by immunofluorescence. (Scale bar, 100 μm.)

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Expressing, shRNA, Cell Culture, Flow Cytometry, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Differentiation of hPSCs to cardiomyocytes via small-molecule modulation of regulatory elements of canonical Wnt signaling. GSK-3 inhibition stimulates mesoderm commitment of undifferentiated hPSCs. In the later stages of differentiation, expression of β-catenin shRNA or inhibition of Wnt ligand production stimulates cardiomyocyte differentiation.

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Inhibition, Expressing, shRNA

Journal: Neuron

Article Title: CONSERVED HIGHER ORDER CHROMATIN REGULATES NMDA RECEPTOR GENE EXPRESSION AND COGNITION

doi: 10.1016/j.neuron.2014.10.032

Figure Lengend Snippet: (A) 500kb of linear genome (mm9, chromosome 6:135,623,529-136,123,529), encompassing Grin2b, including TSS (green), proximal intronic sequences targeted by SETDB1 (yellow), and two loop loopings (red) (1) Grin2bTSS+378kb and (2) Grin2bTSS+471kb, with sequences homologue to human GRIN2BTSS+348kb and GRIN2BTSS+449kb (Fig. S1) (B) (Top) Browser tracks for histone marks H3K4me3 and H3K27ac in adult mouse cerebral cortex(Dixon et al., 2012). (C) Fold-change (CK-Setdb1 transgenic (Tg) /wildtype littermate (Wt) of 3C PCR from adult Tg and Wt cortex (see also Fig. S2). Notice increased physical interactions of (yellow) TSS-bound +15 to +40kb intronic sequence, and significant decrease in (red box no. 1) Grin2bTSS+378kb and (red box no. 2) Grin2bTSS+471kb. Two-way ANOVA, 3C interaction × genotype F(7,32)=54.905(p<0.001). Newman-Keuls post-hoc *, **; P< 0.05, 0.01. (D) Quantification of Grin2b (left) RNA and (right) protein in adult (6–8 week) CK-Setdb1 Tg and wildtype (Wt) littermate control cortex. mean ± S.D, N=5 (RNA) and N=3 (immunoblot)/group. (E) Activity-dependent regulation of Grin2b higher order chromatin in hippocampal neurons. (top, left to right) Grin2b RNA, 3C quantification of Grin2bTSS+378kb and Grin2bTSS+471kb and SETDB1 occupancy across four regulatory sequences at Grin2b locus. Note significant increase at SETDB1 target site after 15 hours of picrotoxin (PTX) or vehicle control (DMSO). N=3–6 experiments/group, data shown as mean ± S.D..(F) (left) Grin2b-TALE-VP64-GFP specifically targets mouse Grin2b loop no. 2 (Grin2bTSS+471kb), 471 kb downstream of TSS. Images show Neuro2A cells and cultured hippocampal neurons expressing GFP-tagged TALE-VP64. RT-PCR for Grin2b and Gapdh control showing specific expression in cultured neurons. (right) anti-TALE-VP64 ChIP in N2A and NG108 cells and primary cortical neurons, expressed (y-axis) as fold change compared to non-transfected condition (N=3/group; (mean ± S.E.M., * Two-way ANOVA cell type × TALE-VP64 binding F(2,12)=4.04, P<0.05, Bonferroni posthoc P < 0.05).RT-PCR Grin2b, Grin2a and Setdb1 RNA levels in neurons transfected with TALE-VP64-EGFP (TALE), compared to mock-transfected neurons (Con). Notice specific TALE-VP64 mediated increase in Grin2b RNA (N = 6 per group, mean ± S.E.M, *P < 0.05, t-test). See also Figures S1 and S2.

Article Snippet: TALE-based designer transcription factor Two Transcription Activator-Like Element DNA-binding proteins (TALE), fused to four tandem copies of the Herpes simplex Viral protein 16, amino acids 437–447, DALDDFDLDML connected with glycine-serine linkers, were targeted to intergenic sequences in the mouse genome, 471 kb downstream of the Grin2b TSS using the TALE tool box kit (Addgene).

Techniques: Transgenic Assay, Sequencing, Control, Western Blot, Activity Assay, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay

Journal: iScience

Article Title: Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons

doi: 10.1016/j.isci.2024.109166

Figure Lengend Snippet:

Article Snippet: Semiquantitative analysis of western blots was performed with the Image Lab 6.0.1 software (Bio-Rad), using as loading controls βIII-tubulin (for soluble and insoluble fractions), Histone H3 (for nuclear fractions) and actin (for total and cytosolic fractions).

Techniques: Recombinant, Gentle, Reverse Transcription, Cell Viability Assay, DC Protein Assay, Western Blot, Sequencing, Software