(A) Nude mice (6 mice/group) were injected subcutaneously 24 hours post total body irradiation (3 Gy) (S.C.) with control lentivirus Ly3 cells or STAT3 shRNA lentivirus Ly3 cells. Statistical analysis was performed with Two-way ANOVA. (B) NOD-SCID mice (6 mice/group) were injected S.C. with control lentivirus Ly3 cells or STAT3 shRNA lentivirus Ly3 cells. (C) NOD-SCID IL2Rnull mice (6 mice/group) were injected S.C. with control lentivirus Ly3 cells or STAT3 shRNA lentivirus Ly3 cells. Statistical analysis was performed with Two-way ANOVA. (D) NOD-SCID IL2Rnull mice (6 mice/group) were injected S.C. with control lentivirus Ly3 cells or STAT3 shRNA #840 lentivirus Ly3 cells. Statistical analysis was performed with Two-way ANOVA.

(A) Ly3 parental cells were plated in the presence of medium containing 10% FBS and treated with STAT3 inhibitors at the indicated concentrations. After 24 or 48 h, phospho-STAT3 level or cell proliferation were analyzed by flow cytometry or MTS assay, respectively. Values represented as bar graphs are the mean of 3 independent experiments with the standard deviation. (B) Ly3 parental tumor-bearing mice were treated intra tumor with vehicle or STATTIC at 3.75 mg/kg every day. Two hours after the last treatment, samples from each group were harvested. Whole-cell lysates were prepared and subjected to Western blot analysis for phospho-STAT3 level detection. Beta-Actin levels served as loading controls.

(A) Control or STAT3 shRNA lentivirus Ly3 cells were grown for 48/72 hours in full or serum free medium. The percentage of viable and dead apoptotic cells was determined by flow cytometry using Annexin V/Propidium Iodide staining. Values represented as bar graphs are the mean of 3 independent experiments with the standard deviation. (B) Control or STAT3 shRNA lentivirus Ly3 and Ly10 cells were plated in the presence of medium containing 10% FBS in 96-well plates and incubated with 4 ng/mL IL-6 or IL-10 for 48 hours. Following this, the percentage of cell proliferation was determined by MTS assay. Values represented as bar graphs are the mean of 3 independent experiments with the standard deviation. (C) After 48 h of culture cells were collected to isolate RNA and real-time PCR was performed to see changes in mRNA levels of STAT3 target genes. Bars represent relative expression values normalized to the beta-actin levels. Values represented as bar graphs are the mean of 3 independent experiments with the standard deviation.

(A, left panel) Tumors from control lentivirus or STAT3 shRNA lentivirus NOD-SCID IL2Rnull mice were harvested 6 days post injection, stained with cleaved Caspase-3 antibody and analyzed by immunofluorescence. Nuclei were stained with Hoechst. (A, right panel) Two separate cohorts of nude mice were injected S.C. with control lentivirus Ly3 cells or STAT3 shRNA lentivirus Ly3 cells. Tumor samples from each group were harvested at the indicated time and when the volume reached the indicated value; whole-cell lysates were prepared and subjected to Western blotting individually or pooled with tumors harvested from the same group in the same time. Beta-Actin levels served as loading controls. The Integrated Density Values (IDVs) were determined by the densitometry software AlphaImager. Values below each band indicate the ratio between the IDV of that band with the IDV of the correspondent Beta-Actin band. The statistical significance was calculated using the normalized IDVs and the statistical analysis was performed with a student’s-t test (unpaired t test with Welch’s correction). (B) Six days post injection tumors from control lentivirus or STAT3 shRNA lentivirus NOD-SCID mice were isolated, pooled, and processed to perform a cytokine array. Signals were quantified by densitometry and normalized to positive controls. The percentage of cytokine secretion in STAT3 shRNA lentivirus is relative to 100% control lentivirus. (C) After 48 h of culture cells were collected to isolate RNA and PCR array was performed to see changes in mRNA levels of inflammatory cytokines and receptors genes. (D) Tumors from control lentivirus or STAT3 shRNA lentivirus NOD-SCID IL2Rnull mice were harvested 6 days post injection, stained with CD31 (left panel) or Gr1 (right panel) antibody and analyzed by immunofluorescence. Nuclei were stained with Hoechst.