Journal: Journal of Cancer

Article Title: CSF2 Overexpression Is Associated with STAT5 Phosphorylation and Poor Prognosis in Patients with Urothelial Carcinoma

doi: 10.7150/jca.14281

Figure Lengend Snippet: Summary of two significantly and differentially expressed genes related to positive regulation of tyrosine phosphorylation of pSTAT5 in the published transcriptome of UBUC (GSE32894).

Article Snippet: The endogenous peroxidase was quenched by saline for 15 minutes and then incubated with primary monoclonal antibodies against CSF2 (1:100, Cat. No. ab77768, rabbit polyclonal, abcam, Cambridge, MA) and phosphor-STAT5 (pSTAT5, Tyr 694/Tyr 699) (1:50, Cat. No. sc-11761, goat polyclonal, Santa Cruz, CA) for an hour.

Techniques: Phospho-proteomics, Cell Differentiation, Expressing, Activity Assay, Binding Assay, Transduction, Migration, Protein Binding

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: RP11-468E2.5 and the JAK/STAT signaling pathway-related genes are negatively correlated in CRC tissues. A, The expression of RP11-468E2.5, STAT5, STAT6 and CCND1 detected by RT-qPCR. B, The correlation between RP11-468E2.5 with STAT5, STAT6 and CCND1 using Pearson correlation analysis. JAK, janus kinase; lncRNA, long non-coding RNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6; CCND1, Cyclin D1; CRC, colorectal cancer. * p < 0.05, compared with the adjacent normal tissues

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Increased positive expression of p-STAT5, p-STAT6 and CCND1 proteins is evident in CRC tissues. a The expression of p-STAT5, p-STAT6 and CCND1 in CRC tissue detected by immunohistochemistry (× 400). b Antibody labeling conditions following p-STAT5 and p-STAT6, with blocking peptide treatment in immunohistochemistry. c Quantitative analysis for positive expression of p-STAT5, p-STAT6 and CCND1. p-STAT5, p-signal transducer and activator of transcription-5; p-STAT6, p-signal transducer and activator of transcription-6; CRC, colorectal cancer; NC, negative control

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Antibody Labeling, Blocking Assay, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Increased protein expression of STAT5 and STAT6 is found in LOVO, SW620, SW480 and HCT116 cell lines. A and B, Western blot analysis of STAT5, p-STAT5 STAT6, p-STAT6 and GAPDH proteins in different cell lines. C, Subcellular localization of p-STAT5/6 in CRC cells detected by immunofluorescence assay. p-STAT6, p-signal transducer and activator of transcription-6; STAT6, signal transducer and activator of transcription-6; p-STAT5, p-signal transducer and activator of transcription-5; STAT5, signal transducer and activator of transcription-5; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. * p < 0.05, compared with RKO cell line; # p < 0.05, compared with LOVO cell line

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Effect of RP11-468E2.5 on the mRNA expression of JAK/STAT signaling pathway- and apoptosis-related genes in HCT116 and SW480 cells. a RP11-468E2.5 expression and mRNA expression of the JAK/STAT signaling pathway-related genes and apoptosis-related genes determined by RT-qPCR in HCT116 cells. b RP11-468E2.5 expression and mRNA expression of the JAK/STAT signaling pathway- and apoptosis-related genes determined by RT-qPCR in SW480 cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; NC, negative control; JAK1, janus kinase 1; STAT3, signal transducer and activator of transcription-3; STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6; Bcl-2, B-cell leukemia/lymphoma 2; CCND1, Cyclin D1. * p < 0.05, compared with the blank group

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Effects of RP11-468E2.5 on the protein expression of JAK/STAT signaling pathway- and apoptosis-related genes in HCT116 and SW480 cells. a and b Western blot analysis of the JAK/STAT signaling pathway- and apoptosis-related proteins in HCT116 cells. c and d Western blot analysis of the JAK/STAT signaling pathway- and apoptosis-related proteins in SW480 cells. NC, negative control; JAK1, janus kinase 1; STAT3, signal transducer and activator of transcription-3; STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6; Bcl-2, B-cell leukemia/lymphoma 2. * p < 0.05, compared with the blank group

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Western Blot, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: RP11-468E2.5 interacts with STAT5 and STAT6. a Interaction among RP11-468E2.5, STAT5 and STAT6 detected by RNA pull-down assay. b Interaction among RP11-468E2.5, STAT5 and STAT6 verified using RIP assay. STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6. * p < 0.05, compared with IgG group

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Pull Down Assay

Journal: Oncology Reports

Article Title: Upregulation of microRNA-126-5p is associated with drug resistance to cytarabine and poor prognosis in AML patients

doi: 10.3892/or.2015.3839

Figure Lengend Snippet: Effects of transfection with the miR-126-5p mimic on the expression of Klotho, Akt, and JAK/STAT kinases. (A) The relative level of the means and the standard error of Klotho/Actin expression is indicated. Cells were incubated for 48 or 72 h. Klotho mRNA expression levels were significantly decreased following transfection of the miR-126-5p mimic. A positive control, the siRNA of klotho , also significantly decreased Klotho mRNA levels. Comparisons between the 3 groups were performed with the Tukey-Kramer test, ** p<0.01 significantly different from the control group. Transfection of the miR-126-5p mimic also significantly decreased expression levels of the Klotho protein (relative expression level: control, 1.00±0.03; miR-126-5p mimic, 0.61±0.13; mean ± standard error, p=0.046, Student's t-test, n=3). This transfection did not affect the expression level of actin. (B) The miR-126-5p mimic or siRNA of Klotho was transfected into the K562 or HK-2 cells. The relative level of the means and the standard error of Klotho/Actin expression is indicated. The cells were incubated for 72 h. Klotho mRNA expression levels were also significantly decreased following transfection of the miR-126-5p mimic. Comparisons between the 3 groups were performed with the Tukey-Kramer test, * p<0.05, *** p<0.001, significantly different from the control group. (C) Western blot analysis of Akt, the phosphorylated forms of Akt at serine 473 (pAkt), JAK2, the phosphorylated forms of JAK2 at tyrosine 1007 (pJAK2), STAT3, the phosphorylated forms of STAT3 at tyrosine 705 (pSTAT3), STAT5, the phosphorylated forms of STAT5 at tyrosine 694 (pSTAT5) and actin. The miR-126-5p mimic or inhibitor was transfected into KG-1 cells. The cells were incubated for 24 h and then administered cytarabin at a dose of 60 nM for 120 h. The PI3K inhibitor, LY294002 was administered at a dose of 10 μ M for 48 h as a positive control for the decline in the phosphorylation of Akt. (D) Western blot analysis of Akt, the phosphorylated forms of Akt at serine 473 (pAkt), and actin. The miR-126-5p mimic was then transfected into KG-1 cells. Transfection of the miR-126-5p mimic significantly increased the expression of the phosphorylated forms of Akt (relative expression level: control, 1.00±0.14; miR-126-5p mimic, 2.22±0.25; mean ± standard error, ** p<0.01, Student's t-test, n=4). This transfection did not affect the expression levels of actin and Akt.

Article Snippet: Rabbit polyclonal antibodies specific for phosphorylated-JAK2, STAT3, phosphorylated-STAT3, STAT5, phosphorylated-STAT5 and Klotho were purchased from GeneTex.

Techniques: Transfection, Expressing, Incubation, Positive Control, Control, Western Blot, Phospho-proteomics

Journal: Oncology Reports

Article Title: Upregulation of microRNA-126-5p is associated with drug resistance to cytarabine and poor prognosis in AML patients

doi: 10.3892/or.2015.3839

Figure Lengend Snippet: Effects of transfection with the miR-126-5p mimic on the expression of Klotho, Akt, and JAK/STAT kinases. (A) The relative level of the means and the standard error of Klotho/Actin expression is indicated. Cells were incubated for 48 or 72 h. Klotho mRNA expression levels were significantly decreased following transfection of the miR-126-5p mimic. A positive control, the siRNA of klotho , also significantly decreased Klotho mRNA levels. Comparisons between the 3 groups were performed with the Tukey-Kramer test, ** p<0.01 significantly different from the control group. Transfection of the miR-126-5p mimic also significantly decreased expression levels of the Klotho protein (relative expression level: control, 1.00±0.03; miR-126-5p mimic, 0.61±0.13; mean ± standard error, p=0.046, Student's t-test, n=3). This transfection did not affect the expression level of actin. (B) The miR-126-5p mimic or siRNA of Klotho was transfected into the K562 or HK-2 cells. The relative level of the means and the standard error of Klotho/Actin expression is indicated. The cells were incubated for 72 h. Klotho mRNA expression levels were also significantly decreased following transfection of the miR-126-5p mimic. Comparisons between the 3 groups were performed with the Tukey-Kramer test, * p<0.05, *** p<0.001, significantly different from the control group. (C) Western blot analysis of Akt, the phosphorylated forms of Akt at serine 473 (pAkt), JAK2, the phosphorylated forms of JAK2 at tyrosine 1007 (pJAK2), STAT3, the phosphorylated forms of STAT3 at tyrosine 705 (pSTAT3), STAT5, the phosphorylated forms of STAT5 at tyrosine 694 (pSTAT5) and actin. The miR-126-5p mimic or inhibitor was transfected into KG-1 cells. The cells were incubated for 24 h and then administered cytarabin at a dose of 60 nM for 120 h. The PI3K inhibitor, LY294002 was administered at a dose of 10 μ M for 48 h as a positive control for the decline in the phosphorylation of Akt. (D) Western blot analysis of Akt, the phosphorylated forms of Akt at serine 473 (pAkt), and actin. The miR-126-5p mimic was then transfected into KG-1 cells. Transfection of the miR-126-5p mimic significantly increased the expression of the phosphorylated forms of Akt (relative expression level: control, 1.00±0.14; miR-126-5p mimic, 2.22±0.25; mean ± standard error, ** p<0.01, Student's t-test, n=4). This transfection did not affect the expression levels of actin and Akt.

Article Snippet: Rabbit polyclonal antibodies specific for phosphorylated-JAK2, STAT3, phosphorylated-STAT3, STAT5, phosphorylated-STAT5 and Klotho were purchased from GeneTex.

Techniques: Transfection, Expressing, Incubation, Positive Control, Control, Western Blot, Phospho-proteomics