Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: mRNA changes after sunitinib treatment in KURC1 and KURC2 xenograft tumors.

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: (A) Evaluation of IL13RA2 mRNA expression in KURC1 and KURC2 xenograft tumors treated with sunitinib or vehicle by qPCR. All samples were prepared in triplicate and data are presented as the mean ± SE from indicated number of samples. Columns, mean; bar, SE. The difference in the mRNA expression levels between the sunitinib-treated group and control or sensitive group in KURC1 was statistically significant (* P < 0.01; Students’ t -test). There was no significant difference in KURC2 groups. (B) Immunohistochemical staining of IL13RA2 in KURC1 xenograft tumors. Scale bar, 50 μm. (C) IL13RA2 expression in human ccRCC tumors with the response to sunitinib treatment evaluated by immunohistochemistry. ccRCC tumor samples were collected from patients prior to sunitinib treatment. Left: representative pictures of immunohistochemistry sections of tumors showing none, weak, or strong staining for IL13RA2. Right: ratio of IL13RA2 expression pattern and correlation of the response to sunitinib treatment. Scale bar, 100 μm.

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Expressing, Control, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: (A) Immunoblot analysis of 786-O subclones infected with retrovirus encoding mock or WT IL13RA2. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes in subcutaneous xenograft tumors from 786-O subclones infected with (B) mock or (C) WT IL13RA2 treated with sunitinib and vehicle (control). Each time point represents the mean ± SE of tumor volume in each group. The difference in tumor size between the treatment group and control was statistically significant in 786-O-mock cells but not statistically significant in 786-O-IL13RA2 cells (* P < 0.05, n.s.: not significant; two-way repeated ANOVA). The horizontal arrow bars indicate the periods of sunitinib administration. (D) Immunoblot analysis of Caki-1 subclones infected with lentivirus encoding scrambled or IL13RA2 shRNA. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes of subcutaneous xenograft tumors from a Caki-1 subclone infected with (E) scrambled or (F) IL13RA2 shRNA treated with sunitinib and vehicle (control). Each time point represents the mean ± SE of tumor volume in each group. Day 0 is the first day of sunitinib administration 4 weeks after transplantation. The difference in tumor size between the treatment group and control was not significant in Caki-1-sh-scrambled cells but statistically significant in Caki-1-sh-IL13RA2 cells (n.s.: not significant, * P < 0.05; two-way repeated ANOVA). The arrow bars indicate the period of sunitinib administration.

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Western Blot, Infection, Control, shRNA, Transplantation Assay

Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: MVD was decreased by sunitinib treatment of each xenograft tumor derived from (A) 786-O or (B) Caki-1 subclones regardless of IL13RA2 expression level. MVD was determined from CD31 staining using Image J software. Statistical analysis was performed using the Students’ t -test (* P < 0.01). Immunoblot analysis of (C) 786-O subclones and (D) Caki-1 subclones. IL13RA2 expression was negatively correlated with the phosphorylation of STAT6. Whole cell extracts were immunoblotted using the indicated antibodies. ssDNA staining of xenograft tumors derived from (E) 786-O subclones and (B) Caki-1 subclones treated with sunitinib or vehicle only. Apoptosis was assessed by calculating the ssDNA positivity rate. Statistical analysis was performed using the Students’ t -test (* P < 0.05, ** P < 0.01).

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Derivative Assay, Expressing, Staining, Software, Western Blot, Phospho-proteomics