Journal: Cancer Research

Article Title: JAK-STAT Blockade Inhibits Tumor Initiation and Clonogenic Recovery of Prostate Cancer Stem-like Cells

doi: 10.1158/0008-5472.can-13-0874

Figure Lengend Snippet: Figure 3. Activation of STAT3 is mediated though IL-6 and JAKs. A, Western blot analysis showing differential STAT3 phosphorylation in a series of primary cultures derived from patients with cancer (samples 4, 15, 19–21) and benign disease (n ¼ 2). The results are expressed as the ratio of pSTAT3 to total STAT3 (tSTAT3). b-Actin was used as a loading control. B, Western blot analysis of a primary culture (HNPC) treated with P6 (0.5 or 5 mmol/L for 16 hours) for pSTAT3 and total STAT3, with b-actin as a loading control. The levels of each were quantified from three primary cultures, derived from patients with HNPC (samples 17, 22, and 23), and are expressed as the ratio of rSTAT3:tSTAT3. C, Western blot analysis of primary cancer cells treated with 10 mg/mL CNTO 328 (þ) or an isotype control () for 2, 4, and 6 days. Right, the ratio of rSTAT3:tSTAT3 from 3 patients with HNPC (samples 22– 24). Gray bar, isotype control; black bar, CNTO 328. Quantification of Western blot analyses was carried out using ImageJ software. Statistical analysis was conducted using the Student t test; , P < 0.05. M, medium only; VC, vehicle control.

Article Snippet: Membranes were blocked with 5% semidried milk for 1 hour and then incubated with primary antibodies against phospho-STAT3 (Tyr705; 1:500), STAT3 (1:1,000; Cell Signaling Technology), and b-actin (1:10,000; Sigma).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Derivative Assay, Control, Software

Journal: Cancer Research

Article Title: JAK-STAT Blockade Inhibits Tumor Initiation and Clonogenic Recovery of Prostate Cancer Stem-like Cells

doi: 10.1158/0008-5472.can-13-0874

Figure Lengend Snippet: Figure 4. STAT3 blockade suppresses colony-forming ability. Primary prostate cells derived from patients with either Gleason grade 7 HNPC (samples 10, 25– 30), Gleason 9 HNPC (samples 11, 23, and 31), or HT [which included hormone-sensitive and castrate-resistant patients (samples 16, 32–35)] were treated for 16 hours with 5 mmol/L P6 (A) or 10 mg/mL CNTO 328 for 6 days (samples 23, 35, and 36; B). CD133þ (stem-like) and CD133 (progenitor) cells were subsequently isolated and plated (at clonal density) in the presence of irradiated STO feeder cells to determine CFE. Colonies were scored if they had undergone five or more population doublings (32 cells), approximately 14 days after plating. The results are expressed as relative to the CFE of the nontreated cells (1%). Each circle represents an individual patient and the bar is the average of each group. C, Western blot analysis of a representative primary culture (sample 37) treated overnight with increasing concentrations of LLL12. D, relative CFE following treatment of stem-like and progenitor cells with 1 mmol/L LLL12 for 24 hours. Primary cultures were derived from patients with Gleason 8/9 disease and included patients who had undergone androgen-ablation therapy (samples 23, 33, 35, and 36). Statistical analyses was conducted using the Student t test; , P < 0.05.

Article Snippet: Membranes were blocked with 5% semidried milk for 1 hour and then incubated with primary antibodies against phospho-STAT3 (Tyr705; 1:500), STAT3 (1:1,000; Cell Signaling Technology), and b-actin (1:10,000; Sigma).

Techniques: Derivative Assay, Isolation, Irradiation, Western Blot