Journal: Bioengineering

Article Title: Formation and Long-Term Culture of hiPSC-Derived Sensory Nerve Organoids Using Microfluidic Devices

doi: 10.3390/bioengineering11080794

Figure Lengend Snippet: Characterization of neurospheres and axon bundles using fluorescent immunostaining. ( a , b ) TUJ1 as a neuron marker and ( a ) ISL1 as a sensory neuron marker. An organoid cultured for 39 days was used. ( b ) TRPV1 as a nociceptor marker. An organoid cultured for 28 days was used. White-colored scale bars: 500 μm. Gray-colored scale bars in close-up panels for axon bundles: 100 μm.

Article Snippet: For molecular characterization, the sensory nerve organoids were labeled with anti-tubulin β3 (TUBB3/TUJ1) mouse monoclonal antibody (MMS-435P, BioLegend, CA, USA), anti-ISL LIM homeobox 1 (ISL1) rabbit polyclonal antibodies (GTX102807, GeneTex, TX, USA), or anti-TRPV1 rabbit polyclonal antibodies (NBP1-97417, Novus Biologicals, CO, USA), followed by incubation with Alexa Fluor-conjugated anti-mouse or anti-rabbit polyclonal secondary antibodies (ab150109, ab150068, Abcam, Waltham, MA, USA).

Techniques: Immunostaining, Marker, Cell Culture

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

doi: 10.31083/j.fbl2903100

Figure Lengend Snippet: Fig. 5. ISL1 modulates neuroactive pathway in subclusters of single cell RNA-seq data and The Cancer Genome Atlas (TCGA)

Article Snippet: After cell counting, the cells were incubated with ISL LIM Homeobox 1 (ISL1) monoclonal antibody (1:200, 15661-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C in the dark for 60 min, followed by incubation for 30 min with a fluorescent secondary antibody (1:200, SA00013-2, CoraLite488-conjugated goat anti-rabbit IgG (H+L)).

Techniques: RNA Sequencing

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

doi: 10.31083/j.fbl2903100

Figure Lengend Snippet: Fig. 6. Flow cytometric sorting of neuroendocrine tumor cells to obtain ISL1 expression group, and explore the differences in cell function in vitro. (A) Flow cytometric sorting of neuroendocrine tumor cell line H446 according to the difference in the expression of

Article Snippet: After cell counting, the cells were incubated with ISL LIM Homeobox 1 (ISL1) monoclonal antibody (1:200, 15661-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C in the dark for 60 min, followed by incubation for 30 min with a fluorescent secondary antibody (1:200, SA00013-2, CoraLite488-conjugated goat anti-rabbit IgG (H+L)).

Techniques: Expressing, Cell Function Assay, In Vitro

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: ISL-1 is highly expressed in the majority subtypes of NHL. (A) Immunohistochemistry for ISL-1 expression in normal lymph nodes and multiple subtypes of lymphoma specimens was performed. Representative images of ISL-1 expression level and cellular distribution in different samples are shown (200 ×). Scale bars = 100 μm. (B) Staining scores of ISL-1 in normal lymph nodes, HL and NHL were statistically analyzed by χ 2 test. (C to D) The mRNA and protein levels of ISL-1 in NHL cell lines and health human peripheral white blood cells (PBC) were analyzed by RT-PCR (C) and Western blot (D) analysis. Numbers 1–7 represent PBC samples from different donors.

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: ISL-1 promotes NHL cells proliferation and affects cell-cycle phase distributions. (A) The relative proliferation rate of stably transfected cells was determined by CCK-8 assay at indicated time post-seeding. (B) Cell cycle distributions were analyzed by flow cytometry. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t -test (* p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01 vs. each control). (Control, ISL-1, Non-silencer and ISL-1 siRNA represent the cells transfected with pcDNA3.1, pcDNA3.1-ISL-1, pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid, respectively)

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Stable Transfection, Transfection, CCK-8 Assay, Flow Cytometry, Plasmid Preparation

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: ISL-1 enhances xenografted lymphoma development in vivo . (A to D) NOD-SCID (nonobese diabetic/severe combined immunodeficient) mice were injected s.c. with different NHL cells that were stably transfected with pcDNA3.1 (Control), or pcDNA3.1-ISL-1 (ISL-1) construct (A,C) , pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid (B,D) . The tumor size was monitored at indicated days post-injection. Statistical analysis was carried out with 2-way ANOVA. (E) The mice were killed after the last measurement of tumor volume, whole-cell lysate of 2 tumor samples of each group were prepared and subjected to Western blot analysis for ISL-1 level detection. GAPDH served as an internal control.

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: In Vivo, Injection, Stable Transfection, Transfection, Construct, Plasmid Preparation, Western Blot

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: The expression of ISL-1 is positively correlated to the expression of p-STAT3, p-c-Jun and c-Myc. (A) NHL cell lines were analyzed by Western blot with indicated antibodies. (B) Immunohistochemistry for ISL-1, p-STAT3, p-c-Jun and c-Myc expression were performed in multiple specimens of normal lymph nodes (top panel) and NHL patients (bottom panel). Representative images of ISL-1, p-STAT3, p-c-Jun and c-Myc expression levels and cellular distributions in different samples are shown (200 ×). Scale bars = 100 μm

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: ISL-1 promotes the expression of c-Myc in NHL cell lines. (A to B) The expression of ISL-1 and c-Myc were analyzed at both mRNA and protein levels by real-time RT-PCR (A) and Western blot (B) in Raji cells with stable ISL-1 overexpression and Ly3 cells with stable ISL-1 knockdown. (C) Consensus binding site (TAAT box) for ISL-1 on the human c-Myc enhancer was analyzed by MatInspector software. The mutant sequences are presented and they were used to construct mutant c-Myc-luc . (D to E) The transcriptional activity of ISL-1 on c-Myc-luc wide type (D) , mutants or deletions (E) was analyzed by luciferase reporter assay in HeLa cells. (“WT”, “M” and “D” represent the plasmid of c-Myc-luc wide type, mutant, or deletion, respectively.). Non, WT and ctrl served as the control in corresponding experiments. (F) ISL-1 recruited on c-Myc promoter was analyzed by ChIP assay. Soluble chromatin was prepared from Ly3 cells followed by immunoprecipitation with the antibody against ISL-1 and the normal IgG served as a control. The DNA extractions were amplified using the primers that covered the ISL-1 binding sites on c-Myc enhancer region by real-time PCR. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD. p values were calculated using a Student t -test (* p < 0.05, ** p < 0.01, # p < 0.05 vs. the control).

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Binding Assay, Software, Mutagenesis, Construct, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: p-c-Jun and p-STAT3 contribute to ISL-1 overexpression in NHL cells. (A to B) real-time RT-PCR (A) and Western blot (B) show the expression changes of ISL-1 in Raji and Ly3 cells after treated with JNK signaling pathway activator (Anisomycin, 15 ng/ml) or inhibitor (SP60012, 10 μM) for 6 h. (C to D) real-time RT-PCR (C) and Western blot (D) show the expression changes of ISL-1 in Ly3 cells after treated with JAK/STAT signaling pathway activator (IL-6, 4 ng/ml) or inhibitor (STATTIC, 6 μM) for 24 h. Each bar represents mean ± SD from three samples. p values were calculated using a Student t -test (* p < 0.05, vs. the control).

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: JNK or JAK/STAT signaling inhibitors inhibit NHL cells proliferation through down-regulating ISL-1 expression. (A) The relative proliferation rate of lymphoma cell lines were measured using CCK-8 analysis after treated with JNK signaling pathway inhibitor (SP60012, 10 μM) or JAK/STAT signaling pathway inhibitor (STATTIC, 6 μM). The cell treated with DMSO were used as the control. (B to C) The effect of SP600125 (10 μM) and STATTIC (6 μM) on ISL-1 and c-Myc expression at both mRNA (B) and protein levels (C) were analyzed by real-time RT-PCR and Western blot. The cells treated with DMSO at different time point were used as the corresponding control. (D) The luciferase activity of c-Myc-luc (wide type or M1) was measured in Ly3 cells with or without ISL-1 transcfection after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. (E) The growth inhibition of Ly3 cells with or without ISL-1 transcfection was measured by CCK-8 analysis after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t -test (* p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01 vs. each control).

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Inhibition

Journal: Molecular Cancer

Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex

doi: 10.1186/1476-4598-13-181

Figure Lengend Snippet: p-STAT3/p-c-Jun/ ISL-1 forms a transcriptional complex and binds directly to ISL-1 promoter. (A) Consensus binding sites for p-STAT3 and p-c-Jun on the ISL-1 promoter were analyzed by Matinspector software. (B) The luciferase activity of ISL-1-luc was analyzed by luciferase reporter assay in Ly3 cells after treated with IL-6 (4 ng/ml), STATTIC (6 μM), Anisomycin (15 ng/ml) or SP600125 (10 μM) for 24 h. (C) ChIP assay was performed with anti-p-STAT3 Ab (left panel) or anti-p-c-Jun Ab (right panel) for immunoprecipitation using chromatin harvested from Ly3 cells. The DNA extractions were amplified using the primers that cover the p-STAT3 (primers 2) or p-c-Jun (primers 4) binding sites, or control primers (primers 1, 3) on the ISL-1 promoter by real-time PCR with normal IgG as a control. (D) Co-IP assay was performed in Ly3 for the transcriptional complex recruited on the ISL-1 promoter. (E) ChIP-re-IP assay was performed first with anti-ISL-1 Ab or rabbit IgG Ab and then with anti-p-STAT3, anti-p-c-Jun or IgG Abs for immunoprecipitation using chromatin harvested from Ly3 cells. (F) The transcriptional activity of ISL-1 on ISL-1-luc was analyzed in Ly3 cells by luciferase reporter assay. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t -test (* p < 0.05, ** p < 0.01 vs. the control).

Article Snippet: Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Binding Assay, Software, Luciferase, Activity Assay, Reporter Assay, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Co-Immunoprecipitation Assay