Journal: International Journal of Oncology

Article Title: Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

doi: 10.3892/ijo.2016.3601

Figure Lengend Snippet: Characteristics of patients with osteosarcoma and non-cancer participants.

Article Snippet: Osteosarcoma cell line (MMNG-HOS) catalog no. CLS 300289 passage 24 and U2OS catalog no. CLS 300364 passage 24 were used as representative osteosarcoma cells.

Techniques: Biomarker Discovery

Journal: International Journal of Oncology

Article Title: Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

doi: 10.3892/ijo.2016.3601

Figure Lengend Snippet: Representative 2D-PAGE of osteosarcoma and osteoblast. (A) Red arrows show spots significantly upregulated in osteosarcomas; green arrows show spots significantly upregulated in osteoblasts. (B) Heat map shows fold changes of % volume of protein spots between osteosarcoma and osteoblast in each matched set.

Article Snippet: Osteosarcoma cell line (MMNG-HOS) catalog no. CLS 300289 passage 24 and U2OS catalog no. CLS 300364 passage 24 were used as representative osteosarcoma cells.

Techniques:

Journal: International Journal of Oncology

Article Title: Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

doi: 10.3892/ijo.2016.3601

Figure Lengend Snippet: Summary of significant altered proteins in primary osteosarcoma cells identified by LC-MS/MS.

Article Snippet: Osteosarcoma cell line (MMNG-HOS) catalog no. CLS 300289 passage 24 and U2OS catalog no. CLS 300364 passage 24 were used as representative osteosarcoma cells.

Techniques:

Journal: International Journal of Oncology

Article Title: Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

doi: 10.3892/ijo.2016.3601

Figure Lengend Snippet: (A) KSRP expression levels in experimental samples (1, pooled sample of osteosarcoma; 2, pool sample of osteoblasts; 3–15 (odd number), individual osteosarcoma cases; 2–16 (even number), individual osteoblast cases; 17, MNNG-HOS; 18, U2OS; 19, HeLa cells as the positive control). (B) KSRP expression in 12 representative biopsy samples of osteosarcoma cases in the separate group.

Article Snippet: Osteosarcoma cell line (MMNG-HOS) catalog no. CLS 300289 passage 24 and U2OS catalog no. CLS 300364 passage 24 were used as representative osteosarcoma cells.

Techniques: Expressing, Positive Control

Journal: International Journal of Oncology

Article Title: Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

doi: 10.3892/ijo.2016.3601

Figure Lengend Snippet: Significantly decreased migratory behavior of osteosarcoma cell lines after KSRP knock-down by siRNA-1 (siR-1) and si-RNA2 (siR-2) compared with non-sense (NS) siRNA as a control (A) at 48 h and (B) at 72 h. (C) Cell growth curve showing significantly decreased proliferative ability of osteosarcoma cell lines after KSRP knock-down.

Article Snippet: Osteosarcoma cell line (MMNG-HOS) catalog no. CLS 300289 passage 24 and U2OS catalog no. CLS 300364 passage 24 were used as representative osteosarcoma cells.

Techniques: Knockdown, Control

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: PloS one

Article Title: Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

doi: 10.1371/journal.pone.0048028

Figure Lengend Snippet: Figure 1. Nkx2-5, Tbx5, Gata4 and Myocd lentiviral vectors transactivated cis-element reporters containing cognate sequences of Nkx2-5, Tbx5, Gata4, and Srf binding sites. CV-1 cells were cotransfected with Tet-transactivator vector, corresponding luciferase reporters, and one or more of Nkx2-5, Tbx5, Gata4, Myocd, or LacZ tetracycline-dependent lentiviral vectors. Dual-luciferase assays were performed 48 hours after doxycycline induction. (A) TRE-Nkx2-5-IPR transactivated NKE cis-element reporter. (B) TRE-Tbx5-INR transactivated TBE cis-element reporter. (C) TRE- Gata4-IPG transactivated GATA cis-element reporter. (D) TRE-Myocd-ING transactivated SRE cis-element reporter in the presence of Srf. Each experiment was performed in quadruplicate. Blank, without the addition of tetracycline-dependent lentiviral vector; TRE-IPR, TRE-INR, TRE-IPG, and TRE-ING are empty vector controls for Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. TRE-LacZ-IPR, TRE-LacZ-INR, TRE-LacZ-IPG, and TRE-LacZ-ING are LacZ vector controls for Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. TRE-N-IPR, TRE-T-INR, TRE-G-IPG, and TRE-M-ING are Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. *P,0.01 vs LacZ groups. doi:10.1371/journal.pone.0048028.g001

Article Snippet: Briefly, complementary deoxyribonucleic acids (cDNAs) of human Nkx2-5 (OriGene) and peroxisome Discosoma sp. red fluorescent protein variant MST (DsRed-MST), human Tbx5 (OriGene) and nuclear DsRed-MST, human Gata4 (OriGene) and peroxisome enhanced green fluorescent protein (EGFP), and human Myocd (accession number AY764180, provided by Dr. Antoine A.F. de Vries) [22] and nuclear EGFP were linked together with an encephalomyocarditis virus-derived IRES sequence, respectively.

Techniques: Binding Assay, Plasmid Preparation, Luciferase

Journal: PloS one

Article Title: Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

doi: 10.1371/journal.pone.0048028

Figure Lengend Snippet: Figure 2. Tbx5+Myocd and Tbx5+Gata4+Myocd combinations induced Myh6 and Tnnt2 cardiac marker protein expression in 10T1/ 2 non-myoblastic cells. 10T1/2 fibroblasts were infected with Nkx2-5, Tbx5, Gata4, Myocd lentivirus alone, or different combinations. LacZ lentivirus was used as control. Ten days after doxycycline induction, Myh6 and Tnnt2 protein expression was examined by incubating the cells with Myh6 or Tnnt2 primary antibody and was visualized by Alexa Fluor 350-conjugated secondary antibody. Only T+M and T+G+M combinations induced de novo Myh6 and Tnnt2 protein expression. Myh6+ and Tnnt2+ cell induction rates in designated cell populations were summarized in Table 1. Insets showed nascent fibril-like structures. Scale bar = 20 mm. N, Nkx2-5; T, Tbx5; G, Gata4; M, Myocd. doi:10.1371/journal.pone.0048028.g002

Article Snippet: Briefly, complementary deoxyribonucleic acids (cDNAs) of human Nkx2-5 (OriGene) and peroxisome Discosoma sp. red fluorescent protein variant MST (DsRed-MST), human Tbx5 (OriGene) and nuclear DsRed-MST, human Gata4 (OriGene) and peroxisome enhanced green fluorescent protein (EGFP), and human Myocd (accession number AY764180, provided by Dr. Antoine A.F. de Vries) [22] and nuclear EGFP were linked together with an encephalomyocarditis virus-derived IRES sequence, respectively.

Techniques: Marker, Expressing, Infection, Control

Journal: PloS one

Article Title: Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

doi: 10.1371/journal.pone.0048028

Figure Lengend Snippet: Figure 3. Tbx5+Gata4+Myocd was the most effective activator of cardiac genes. 10T1/2 fibroblasts were infected with Tbx5, Gata4, Myocd lentivirus alone, or different combinations. LacZ lentivirus was used as control. Genetically unbiased genome-wide expression profiles were obtained 14 days after doxycycline induction. (A) Global gene expression. (B) Cardiac structural gene enrichment. (C) Smooth muscle gene enrichment. (D) Skeletal muscle gene enrichment. (E) Cardiac-related transcription factor enrichment. Signal values were log2 transformed. Signal values below 0 were set to 1. Each group included biological triplicates. T, Tbx5; G, Gata4; M, Myocd; TG, T+G; GM, G+M; TM, T+M; TGM, T+G+M. doi:10.1371/journal.pone.0048028.g003

Article Snippet: Briefly, complementary deoxyribonucleic acids (cDNAs) of human Nkx2-5 (OriGene) and peroxisome Discosoma sp. red fluorescent protein variant MST (DsRed-MST), human Tbx5 (OriGene) and nuclear DsRed-MST, human Gata4 (OriGene) and peroxisome enhanced green fluorescent protein (EGFP), and human Myocd (accession number AY764180, provided by Dr. Antoine A.F. de Vries) [22] and nuclear EGFP were linked together with an encephalomyocarditis virus-derived IRES sequence, respectively.

Techniques: Infection, Control, Genome Wide, Expressing, Gene Expression, Transformation Assay

Journal: iScience

Article Title: A small-molecule Skp1 inhibitor elicits cell death by p53-dependent mechanism

doi: 10.1016/j.isci.2022.104591

Figure Lengend Snippet:

Article Snippet: β-TRCP , Santa Cruz , Cat# sc-166492; RRID: AB_2102598.

Techniques: Control, Virus, Recombinant, Synthesized, Protease Inhibitor, Transfection, Marker, Membrane, Caspase-Glo Assay, Protein Extraction, Bicinchoninic Acid Protein Assay, Western Blot, Plasmid Preparation, SYBR Green Assay, 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: Fig. 2 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, Expressing, Software