Journal: Cells

Article Title: The Warburg Trap: A Novel Therapeutic Approach for Targeting Osteosarcoma.

doi: 10.3390/cells13010061

Figure Lengend Snippet: Figure 2. Warburg Trap drugs significantly reduce the colony-forming capacity of OS cells. OS cell lines (n = 6) were seeded at low density in culture plates and treated with the WT drugs (Metformin (1 mM), Monensin (5 nM), and Fenofibrate (10 µM)) in an acidic culture medium (pH 6.5). After 24 h and 48 h of treatment, the culture medium was replaced by a normal cell culture medium with a physiological pH value of 7.4. Colonies that formed during a further seven days of culture were stained with haematoxylin and photographed before (A) the number of colonies and (B) the size of the colonies were quantified using the software ImageJ version 1.48v (* p < 0.01 compared to untreated controls). (C) Representative photographs of stained colonies obtained with cell line 143B analysed in duplicates. (D) Analysis of colony numbers that formed after pre-treatment of osteosarcoma cell lines (n = 6) with WT drugs and subsequent cultivation for 14 days in soft agar (* p < 0.01 compared to untreated controls).

Article Snippet: The following six commercially available human osteosarcoma cell lines were used in this study: HOS (American Tissue Culture Collection, Rockville, MD, USA), HOS 143B (Sigma-Aldrich, Taufkirchen, Germany), CAL-72 (DSMZ, Braunschweig, Germany), MNNG-HOS, SaOS-2, and U2OS (Cell Line Service GmbH, Eppelheim, Germany).

Techniques: Cell Culture, Staining, Software

Journal: Cells

Article Title: The Warburg Trap: A Novel Therapeutic Approach for Targeting Osteosarcoma.

doi: 10.3390/cells13010061

Figure Lengend Snippet: Figure 3. DDIT3-mediated downregulation of WNT target genes by WT drugs. Osteosarcoma cell lines (n = 3) were treated with WT drugs (Metformin (1 mM), Monensin (5 nM), and Fenofibrate (10 µM)) (MeMoFe) for the indicated time before the expression of DDIT3 and the WNT target genes AXIN2, CCND1 and LGR5 were analysed by real-time quantitative RT-PCR analysis. The expression of the reference gene RPS13 (ribosomal protein S13) was used for normalisation. Data are presented as percent of untreated 24 h controls (* p < 0.05 compared to untreated controls).

Article Snippet: The following six commercially available human osteosarcoma cell lines were used in this study: HOS (American Tissue Culture Collection, Rockville, MD, USA), HOS 143B (Sigma-Aldrich, Taufkirchen, Germany), CAL-72 (DSMZ, Braunschweig, Germany), MNNG-HOS, SaOS-2, and U2OS (Cell Line Service GmbH, Eppelheim, Germany).

Techniques: Expressing, Quantitative RT-PCR

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) WWC1-3 ubiquitination. HEK293A cells were cotransfected with FLAG-tagged WWC1-3 and hemagglutinin (HA)–tagged ubiquitin, immunoprecipitated WWC proteins were subjected to immunoblotting using HA antibody. Cells were treated with bortezomib (BTZ) for 6 hours before IP assay. ( B ) Schematic diagram of Bio-ID and mass spectrometry procedures. ( C ) Known interacting proteins and identified E3 ligases for WWC1. ( D ) Elevated protein levels of WWC1-3 in BTRC and BTRCP2 dKO HEK293A cells, with quantification shown. Asterisks indicate nonspecific bands. ( E ) Decreased WWC1-3 levels in HEK293A cells with ectopic β-TrCP1 or β-TrCP2. ( F ) WWC1-3 interact with β-TrCP1/2. HA-tagged WWC1/2/3 were cotransfected with FLAG-tagged β-TrCP1/2 into HEK293A cells, respectively, and subjected to coimmunoprecipitation (co-IP) assay. ( G ) Increased ubiquitination of WWC1 in cells with ectopic expression of β-TrCP1 and/or β-TrCP2. MYC-β-TrCP1 and β-TrCP2 were cotransfected with FLAG-WWC1 and HA-ubiquitin (Ub). WWC1 was immunoprecipitated, and ubiquitination was detected by HA antibody. Cells were treated with BTZ for 6 hours before IP assay. ( H ) Amino acids 601 to 700 of WWC1 are essential for interaction with β-TrCP1. WT or truncated WWC1 was cotransfected with HA-tagged β-TrCP1 and subjected to co-IP assay. ( I ) S631 is essential for WWC1 to interact with β-TrCP1/2. WT or S631A FLAG-WWC1 was cotransfected with HA-β-TrCP2 or MYC-β-TrCP1 and subjected to co-IP assay. ( J ) S631A mutant WWC1 shows impaired ubiquitination. WT or S631A FLAG-WWC1 was cotransfected with indicated plasmids into HEK293A cells, WWC1 was immunoprecipitated, and ubiquitination was determined. Cells were treated with BTZ for 6 hours before IP assay. ( K ) S631A mutant WWC1 is more stable. WT or S631A WWC1 was overexpressed in HEK293A cells, treated with CHX (100 μg/ml) for 4 to 12 hours, and whole-cell lysate was collected for immunoblotting, and protein levels of WWC1 were quantified.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Mass Spectrometry, Co-Immunoprecipitation Assay, Expressing, Mutagenesis

Journal: Science Advances

Article Title: WWC proteins–mediated compensatory mechanism restricts schwannomatosis driven by NF2 loss of function

doi: 10.1126/sciadv.adp4765

Figure Lengend Snippet: ( A ) Reduced ubiquitination of WWC1/2 in NF2 KO cells. WT and NF2 KO HEK293A cells were cotransfected with FLAG-WWC1/2 and HA-Ub, WWC1/2 were immunoprecipitated, and ubiquitination were determined by immunoblotting. Cells were treated with BTZ for 6 hours before IP assay. ( B and C ) Concurrent deletion of β-TrCP1 and β-TrCP2 induces expression of WWC1-3 in WT but not NF2 KO cells. Two sets of small guide RNAs targeting BTRC and BTRCP2 and Cas9 plasmids were stably expressed in WT or NF2 KO HEK293A cells, and whole-cell lysate was collected for immunoblotting. WWC1-3 protein levels were also quantified. The asterisk indicates nonspecific bands. ( D ) NF2 interacts with β-TrCP1 and β-TrCP2. FLAG–β-TrCP1 and β-TrCP2 were expressed in HEK293A cells and immunoprecipitated, and co-immunoprecipitated endogenous NF2 was determined by immunoblotting. ( E ) NF2 mediates interaction between WWC1 and β-TrCP1/2. WT and NF2 KO HEK293A cells were cotransfected with indicated plasmids, WWC1 was immunoprecipitated, and bound β-TrCP1/2 was analyzed by immunoblotting and quantified. ( F ) Reduced interaction between WWC1 and β-TrCP2 in NF2 KO MDCK cells. PLA was used to detect the interaction between endogenous WWC1 and β-TrCP2. Maximum intensity projection from z-stack confocal images was used to show PLA spots (red); the nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole). At least 200 cells from 20 microscopic fields per sample were used for quantification. ( G ) S631 is required for the regulation of WWC1 stability by NF2. WT, S631A, or WW domain–deleted (WW del) WWC1 was stably expressed with NF2 in NF2 ; WWC1-3 4KO HEK293A cells. The protein levels of WWC1 were quantified. ( H ) A schematic diagram shows the working mechanism underlying NF2-mediated regulation of WWC1-3 by recruiting β-TrCP1/2. NF2 acts as a platform to recruit WWC1-3 and β-TrCP1/2 together and promote WWC1-3 ubiquitination and degradation through proteasome.

Article Snippet: Primary antibodies specific to WWC1 and β-TrCP2 (13149-1-AP, Proteintech) were used for detecting WWC1 and β-TrCP2 interaction.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Stable Transfection, Staining

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: Cytotoxicity of CBD-enriched hemp oil on normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells. Control cells were incubated with complete cell culture medium (represented as 0 µg CBD/mL). Treated cell viability was expressed as a percentage of control cells’ viability. Data were represented as means ± standard error of the mean. * p < 0.05, ** p < 0.01 vs. NHDF cells (one-way ANOVA: MeWo p = 0.0052, HeLa p = 0.0149, HepG2 p = 0.0400, HOS p = 0.0065).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Incubation, Cell Culture

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: Morphological analysis of normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells treated with CBD-enriched hemp oil at 5, 10, 15, 20 and 25 µg CBD/mL for 48 h. Control cells were incubated with complete cell culture medium (represented as 0 µg CBD/mL).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Incubation, Cell Culture

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: AO/EB staining of normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells treated with CBD-enriched hemp oil at 5, 10, 15, 20 and 25 µg CBD/mL for 48 h. Control cells were incubated with complete cell culture medium (represented as 0 µg CBD/mL).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Staining, Incubation, Cell Culture

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: BAX/BCL2 gene expression ratio in normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells treated with cell line-specific IC 50 of CBD-enriched hemp oil for 48 h. Control cells were incubated with complete cell culture medium. Experiments were done in triplicate, obtained data were expressed as mean 2 −ΔΔCq values (vs. 18S of control cells) ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control cells (NHDF p < 0.0001, MeWo p = 0.013391, HeLa p = 0.049447, HepG2 p = 0.000092, HOS p = 0.002741).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Expressing, Incubation, Cell Culture

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: ( a ) TP53 gene expression; ( b ) MDM2 gene expression in normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells treated with cell line-specific IC 50 of CBD-enriched hemp oil for 48 h. Control cells were incubated with complete cell culture medium. Experiments were done in triplicate, obtained data were expressed as mean 2 −ΔΔCq values (vs. 18S of control cells) ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control cells (TP53: NHDF p = 0.001014, MeWo p = 0.000003, HeLa p = 0.000669, HepG2 p = 0.001736, HOS p = 0.000011; MDM2: NHDF p = 0.065319, MeWo p = 0.001181, HeLa p = 0.002125, HepG2 p = 0.000358, HOS p = 0.033260).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Expressing, Incubation, Cell Culture

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: Intracellular H 2 O 2 production in normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells treated with cell line-specific IC 50 doses of CBD-enriched hemp oil for 24 h. Control cells were incubated with complete cell culture medium. Experiments were done in triplicate, obtained data were expressed as relative fluorescence units (RFU)/mg protein, expressed as fold change of control values and represented as means ± standard error of the mean. * p < 0.05 vs. control cells (NHDF p = 0.058216, MeWo p = 0.160015, HeLa p = 0.011617, HepG2 p = 0.020345, HOS p = 0.055959).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Incubation, Cell Culture, Fluorescence

Journal: Antioxidants

Article Title: New Insights on Hemp Oil Enriched in Cannabidiol: Decarboxylation, Antioxidant Properties and In Vitro Anticancer Effect

doi: 10.3390/antiox10050738

Figure Lengend Snippet: Gene expression of: ( a ) SOD1; ( b ) CAT; ( c ) GPX1; ( d ) GSR in normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells treated with cell line-specific IC 50 of CBD-enriched hemp oil for 48 h. Control cells were incubated with complete cell culture medium. Experiments were done in triplicate, obtained data were expressed as mean 2 −ΔΔCq values (vs. 18S of control cells) ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control cells (SOD1: NHDF p = 0.003067, MeWo p = 0.001484, HeLa p < 0.000001, HepG2 p = 0.001186, HOS p = 0.796912; CAT: NHDF p = 0.002620, MeWo p = 0.000004, HeLa p < 0.000001, HepG2 p = 0.000146, HOS p = 0.821580; GPX1: NHDF p = 0.000002, MeWo p = 0.000027, HeLa p = 0.057635, HepG2 p = 0.337276, HOS p = 0.267738; GSR: NHDF p = 0.019436, MeWo p < 0.000001, HeLa p = 0.001320, HepG2 p = 0.026340, HOS p = 0.006656).

Article Snippet: Normal dermal fibroblasts (NHDF, purchased from PromoCell, Heidelberg, Germany), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells (all malignant cell lines purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were grown in alpha-MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).

Techniques: Expressing, Incubation, Cell Culture

Journal: International Journal of Oncology

Article Title: Protein profiling of osteosarcoma tissue and soft callus unveils activation of the unfolded protein response pathway

doi: 10.3892/ijo.2019.4737

Figure Lengend Snippet: Expression of unfolded protein response-related proteins is upregulated in osteosarcoma cells. Immunoblots of GRP78, GRP94, calreticulin and prelamin-A/C in (A) osteosarcoma cell lines (MNNG/HOS, 143B, U2OS, Saos-2 and MG-63) compared with the osteoblastic cells (hFOB 1.19), and (B) primary osteosarcoma cells (OSP1-6) compared with primary osteoblastic cells (OB1-5). (C) Reverse transcription-quantitative polymerase chain reaction analysis of spliced XBP1 expression levels in osteoblastic cell line vs. osteosarcoma cell lines and (D) primary osteoblastic cells vs. primary osteosarcoma cells. Data are presented as means ± standard deviation from three independent experiments. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 compared to osteoblasts. GRP78, 78 kDa glucose-related protein; GRP94, endoplasmin; OSP, primary osteosarcoma cell; OB, primary osteoblastic cell; XBP1, X-box binding protein 1.

Article Snippet: Osteosarcoma and osteoblastic cell lines used in this study include MNNG/HOS (CLS 300289; Cell Lines Service, GmbH, Eppelheim, Germany), U2OS (CLS-300364; Cell Lines Service, GmbH), 143B (CRL-8303; ATCC, Manassas, VA, USA), MG-63 (CRL-1427; ATCC), Saos-2 (HTB-85; ATCC) and normal human osteoblast cell line hFOB1.19 (CRL-11372; ATCC).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay

Journal: Nature Communications

Article Title: Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis

doi: 10.1038/s41467-018-07333-4

Figure Lengend Snippet: Single-cell RNA-seq identified subpopulations of cardiomyocytes. a Late stage cardiomyocytes (4,689 cells with TNNT2 and ACTC1 expression from day 14 and day 45) were further resolved using ICGS to identify subpopulations of cardiomyocytes. Associated t-SNE cell populations were colored by day of differentiation, as well as by cluster identified (markers within each cluster beside each cluster). b Six cardiomyocyte populations were identified representing subpopulations of cardiomyocytes in early proliferative stages (Cluster 4, 5) expressing cyclins, mid-cardiomyocyte stage (Cluster 2, 3) expressing HAND1 , and more mature cardiomyocytes (Cluster 0, 1) expressing sarcomeric ( MYH6, MYL2, TNNT2, MYBPC3 ) and calcium handling genes ( RYR2, PLN ). c Single cells from day 30 of differentiation were profiled using an independent technology (Fluidigm C1) to resolve coincident mid-to-late state differentiation heterogeneity. Genes associated with distinct observed cell populations identified by the algorithm MarkerFinder are shown. Factors with defining expression in each of the HOPACH clustered populations are shown ( SF1, ISL1, JARID2, TBX5, MYL2/HEY2 , and HOPX) . Two major groups in hiPSC-CMs were observed to express either NR2F2 or MYL2/HEY2 . Within each group, identified heart developmental regulators were identified using the software GO-Elite (left panel)

Article Snippet: To overexpress NR2F2 , TBX5 , and HEY2 in hiPSC-CMs, lentiviral particles were purchased for mGFP-tagged NR2F2 (Cat# RC206753L2V), TBX5 (Cat# RC216520L2V), HEY2 (Cat# RC202544L2V), and control GFP Lent-ORF particles (Cat# PS100071V5) from Origene.

Techniques: RNA Sequencing Assay, Expressing, Software

Journal: Nature Communications

Article Title: Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis

doi: 10.1038/s41467-018-07333-4

Figure Lengend Snippet: Transcriptome interactions between key transcription factors that regulate hiPSC-CM populations. ChIP-seq was performed on transcription factors ( NR2F2, TBX5 , and HEY2 ) enriched within three hiPSC-CM populations. a ChIP-seq peak density demonstrates enrichment of the ChIP-seq signal around the transcriptional start sites (TSS). b GO biological functions were graphed corresponding to peaks called within each ChIP-seq dataset (GREAT analysis, hypergeometric test, p < 0.05). NR2F2 was highly enriched for terms within the nervous system, TBX5 for cardiac tissue, and HEY2 for neural and enteric terms. c Venn diagram demonstrates the number of unique and common genes called (using HOMER) within each ChIP-seq dataset. d Coverage of the reads demonstrating a peak near the MYL 2 locus in the TBX5 and HEY2 ChIP-seq datasets, but not the NR2F2 dataset. e RNA-seq was performed on cardiomyocytes from each gene-edited line [ NR2F2 GE ( NR2F2 GE1 and NR2F2 GE2 lines), HEY2 GE ( HEY2 GE1 and HEY2 GE2 ), and TBX5 GE ( TBX5 GE1 and TBX5 GE2 )], as well as cardiomyocytes over expressing NR2F2 (NR2F2 OE ), TBX5 (TBX5 OE ) and HEY2 (HEY2 OE ). Biological terms associated with genes significantly different (GO-Elite algorithm AltAnalyze) between these lines were annotated (left panel–blue text), as well as the corresponding ChIP-seq peaks identified (right panel). Gene sets associated with each of the single-cell populations from Fig. are highlighted in orange. Genes associated with the enriched GO terms and single-cell populations are displayed to the right of the heatmap. f GO biological analysis was performed on significant genes upregulated or downregulated in hiPSC-CMs overexpressing NR2F2, TBX5 , or HEY2 with corresponding NR2F2, TBX5 , or HEY2 gene-edited lines. g Interactions among genes regulated by NR2F2, TBX5 , and HEY2 were visualized by combining the significant genes called within each NR2F2 GE1 , TBX5 GE1 , and HEY2 GE1 with genes called within each ChIP-seq dataset. Nodes (blue circles) represent individual genes and the links represent the genes that interact with each transcription factor. Red indicates an upregulation and green represents a downregulation observed in the RNA-seq of each gene-edited line. h ChIP-seq/RNA-seq interactions using a selected set of cardiac-specific genes. MYL2 is highlighted to be positively regulated by TBX5 and HEY2 expression

Article Snippet: To overexpress NR2F2 , TBX5 , and HEY2 in hiPSC-CMs, lentiviral particles were purchased for mGFP-tagged NR2F2 (Cat# RC206753L2V), TBX5 (Cat# RC216520L2V), HEY2 (Cat# RC202544L2V), and control GFP Lent-ORF particles (Cat# PS100071V5) from Origene.

Techniques: ChIP-sequencing, RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis

doi: 10.1038/s41467-018-07333-4

Figure Lengend Snippet: NR2F2, TBX5 , and HEY2 influence the electrophysiological properties of hiPSC-CM lines. a Representative traces of calcium imaging on hiPSC-CMs cultured for 14, 30, or 90 days in culture; over time, mature hiPSC-CMs presented a higher baseline ratio, peak ratio amplitude, maximum rising rate and lower transient duration 50. b Calcium imaging was performed on control (day 30-40) and each gene-edited line (day 30–40) to assess the calcium handling ability when NR2F2 , TBX5 , and HEY2 were disrupted. In comparison to control hiPSC-CMs, HEY2 lines displayed a significantly lower maximum rising rate and amplitude. c Representative patch clamp electrophysiological recordings for each gene-edited line. d Action potential parameters for 22 control, 35 NR2F2 GE , 36 TBX5 GE , and 34 HEY2 GE hiPSC-CMs at day 30-40 were quantified for either ventricular-like or atrial-like action potentials. Significantly lower maximum rising rate (dv/dt max ), maximum diastolic potential (MDP), and action potential amplitude (APA) values were observed in the HEY2 GE line when compared to control lines. Increase in action potential amplitude (APD) 50 and APD90 was observed in the ventricular-like NR2F2 GE line. e The proportions of atrial-like, ventricular-like, and nodal-like action potentials were quantified within each gene-edited line. Slightly more ventricular-like action potentials were found in the NR2F2 GE line, whereas a large proportion of atrial-like action potentials were observed in the HEY2 GE line. Lines between bars represent p < 0.05 between groups using a one-way ANOVA

Article Snippet: To overexpress NR2F2 , TBX5 , and HEY2 in hiPSC-CMs, lentiviral particles were purchased for mGFP-tagged NR2F2 (Cat# RC206753L2V), TBX5 (Cat# RC216520L2V), HEY2 (Cat# RC202544L2V), and control GFP Lent-ORF particles (Cat# PS100071V5) from Origene.

Techniques: Imaging, Cell Culture, Patch Clamp