Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: Immunohistochemical expression and survival analysis for the expression of interleukin4 receptor α (IL4Rα) and interleukin13 receptor α1 (IL13Rα1) in clear cell renal cell carcinoma (CCRCC). ( A ) Immunohistochemical expression of IL4Rα and IL13Rα1 in CCRCC tissue. Original magnification, ×400. ( B ) Analysis of sensitivity and specificity of the immunohistochemical staining score of IL4Rα and IL13Rα1 in CCRCC for the event of cancer-specific survival (death of the patient by CCRCC) by receiver operator characteristic curves. The cutoff points were determined at the highest area under the curve (AUC). Red arrow indicates a cutoff point for the IL4Rα immunostaining and blue arrowhead indicates a cut-off point for the IL13Rα1 immunostaining. ( C ) Kaplan-Meier survival analysis for cancer-specific survival and relapse-free survival according to the immunohistochemical positivity for IL4Rα and IL13Rα1 in 199 CCRCC. ( D ) Kaplan-Meier survival analysis in IL4Rα−/IL13Rα1−, IL4Rα−/IL13Rα1+, IL4Rα+/IL13Rα1−, and IL4Rα+/IL13Rα1+ subgroups according to the positivity for IL4Rα and IL13Rα1 expressions. ( E ) Kaplan-Meier survival analysis in two subgroups according to the co-expression patterns of IL4Rα and IL13Rα1; favorable (IL4Rα−/IL13Rα1−, IL4Rα−/IL13Rα1+, or IL4Rα+/IL13Rα1−) and poor prognostic (IL4Rα+/IL13Rα1+) subgroups. 5y-cancer-specific survival (CSS); 5-year cancer-specific survival rate, 10y-CSS; 10-year cancer-specific survival rate; 5y-RFS; five-year relapse-free survival rate, 10y- relapse-free survival (RFS); 10-year relapse-free survival rate.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques: Immunohistochemical staining, Expressing, Staining, Immunostaining

Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: Clinicopathologic variables and the expression of interleukin4 receptor α (IL4Rα) and interleukin13 receptor α1 (IL13Rα1) in clear cell renal cell carcinoma (CCRCC) patients.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques: Expressing

Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: Univariate Cox regression analysis of cancer-specific survival (CSS) and relapse-free survival (RFS) in clear cell renal cell carcinoma (CCRCC) patients.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques:

Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: Multivariate Cox regression analysis of CSS and RFS in CCRCC patients.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques:

Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: Anti-cancer effect by transfection of siRNA against IL4Rα or IL13Rα1 in A498 and ACHN cells. Time-dependent anti-cancer effect by transfection of siRNA against IL4Rα or IL13Rα1 in A498 and ACHN cells for 24, 48, and 72 h incubation after transfection. Cell viability and proliferation rate was determined by WST-1 assay ( A ) and cell counting assay ( B ), respectively. This result is representative data from three biological replicates, and the error bar indicates standard error (STE). * indicates the p -value < 0.05. ( C ) Anti-colony formation ability by transfection of siRNA against IL4Rα or IL13Rα1 in A498 and ACHN cells was determined by colony formation assay for 14 days after transfection. These results are representative data from three biological replicates. Apoptosis in A498 and ACHN cells transfected with siRNA against IL4Rα or IL13Rα1 for 48 h after transfection was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay ( D ) and Annexin V staining analysis ( E ). Cell cycle arrest was determined by cell cycle analysis ( F ). This result is representative data from three biological replicates. ( G ) Western blotting analysis of proteins related to apoptosis and cell cycle arrest in A498 and ACHN cells transfected with siRNA against IL4Rα or IL13Rα1 for 48 h after transfection. β-actin was used for a gel-loading control. Magnification for ( D ): ×20.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques: Transfection, Incubation, WST-1 Assay, Cell Counting, Colony Assay, TUNEL Assay, Staining, Cell Cycle Assay, Western Blot, Control

Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: Interaction between Janus kinase 2 (JAK2) and Forkhead box O3 (FOXO3) protein in A498 and ACHN. ( A ) Silencing IL4Rα in A498 and ACHN cells reduced the interaction between JAK2 and FOXO3. Cells were transfected with control siRNA or siRNA against IL4Rα, and then cell lysates were immunoprecipitated with the anti-pJAK2 antibody. The immunoprecipitated proteins were resolved on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted by anti-IL4Rα, IL13Rα1, FOXO3, and pJAK2 antibody. The light chain of IgG was used for the loading control. ( B ) 293T cells were co-transfected with HA- JAK2 and Flag-FOXO3 (O.E.) or a control plasmid DNA (pCMV3-C-HA and pECE, Con.) as indicated. Then cell lysates were immunoprecipitated with anti-Flag, FOXO3, HA, JAK2, or pTyr antibody. The immunoprecipitated proteins were resolved on the SDS-PAGE and immunoblotted by the indicated antibody, respectively. The light chain of IgG and Coomassie Blue staining of SDS-PAGE were used for the loading control. ( C ) Silencing JAK2 in A498 and ACHN cells increased the expression of FOXO3. Cells were transfected with control siRNA or siRNA against JAK2, and then cell lysates were resolved on the SDS-PAGE and immunoblotted by anti-FOXO3 and JAK2 antibody. β-actin was used for the loading control. ( D ) Pharmacological inhibition of JAK2 in A498 and ACHN cells increased the expression of FOXO3. Cells were treated with dimethyl sulfoxide (DMSO) vehicle control or the indicated concentration of AZD1480, and then cell lysates were resolved on the SDS-PAGE and immunoblotted by anti-FOXO3, pJAK2, and JAK2 antibody. β-actin was used for the loading control.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques: Transfection, Control, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Plasmid Preparation, Staining, Expressing, Inhibition, Concentration Assay

Journal: Cancers

Article Title: Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways

doi: 10.3390/cancers11091394

Figure Lengend Snippet: A diagram describes the role of IL4R complex (a heterodimeric complex of IL4Rα and IL13Rα1) in promoting RCC tumorigenesis via phosphorylation of tyrosine residue in FOXO3 by activation of JAK2 leading to loss of the tumor-suppressive transcriptional activity.

Article Snippet: After incubation, cells were transfected with siRNAs (control (Cat. No.: sc37007), IL4Rα (Cat. No.: sc-35661), IL13Rα1 (Cat. No.: sc-63337), or JAK2 (Cat. No.: sc-39099), 1 nM (Santa Cruz Biotechnology)) or plasmid DNAs (pECE empty/Flag-FOXO3 or pCMV3-C-HA empty/HA-JAK2 plasmid DNA, 1 μg) to 3 μL of Lipofectamine 2000 (Invitrogen) in 300 μL of serum-free media for 6 h at 37 °C in a humidified incubator containing 5% CO 2 in air.

Techniques: Phospho-proteomics, Residue, Activation Assay, Activity Assay