Effects of LY5 are not mediated via STAT3 inhibition.

<p>(A) Antitumor activity of LY5 against OS-1 PDX model of OS. Left panel, growth of individual control (untreated OS-1 xenografts); Center panel, growth of individual OS-1 xenografts in mice receiving LY5 (40 mg/kg twice daily for 5 days/week for 4 consecutive weeks); Right panel, median relative tumor growth (RTV) from control and treated groups. (B) Sarcoma cell lines (RMS cell line RH30 and ES cell lines ES-3 and ES-7) were transfected with either negative control siRNA (NC siRNA) or STAT3-targeting siRNA #7. After 72 hours of transfection, cells were harvested and analyzed for total (T) STAT3 and GAPDH protein expression by immunoblotting. (C) RH30, ES-7, and EW-8 cell lines were transfected with either NC siRNA or STAT3-targeting siRNA. After 48 hrs, cells were incubated with LY5 (1μM) for an additional 48 hrs. Cell viability was then determined using Alamar Blue staining. Data represent the mean ± standard deviation, n = 3. * denotes p < 0.05 by ANOVA.</p

LY5 inhibits pSTAT3 in sarcoma cell lines.

<p>(A) The tumor cell lines RH30, EW8, and RD were treated with increasing concentrations of LY5 for 30 minutes. Cell lysates were collected for Western blotting of p-STAT3, total STAT3, and GAPDH. GAPDH was used as loading control. (B-C) The SJSA cell line was serum-starved overnight and then left untreated or treated with LY5 for two hours followed by stimulation with 50 ng/mL of OSM, IL-6, IFN-γ, IFN-α, or IL-4 for 30 minutes prior to collection of cell lysates for Western blotting of p-STAT3, total STAT3 p-STAT1, total STAT1, p-STAT2, p-STAT4, or p-STAT6. GAPDH was used as loading control.</p

LY5 treatment did not inhibit RMS or OS xenograft tumor growth.

<p>LY5 treatment did not inhibit RMS or OS xenograft tumor growth.</p

Biologic activity of LY5 in sarcoma cell lines.

<p>Dose response curves for LY5: RMS cell lines (RH30, RD, JR-1), ES cell lines (ES-3, ES-6, ES-7, ES-8), and an OS cell line (OS-17) were treated with LY5 (concentrations ranging from 10 nM to 10 μM) for 96 hours. Cell viability was determined using Alamar Blue staining. IC₅₀ values are indicated for each cell line. Analyses were performed in triplicate and data are plotted as means ± standard deviation.</p

LY5 inhibits STAT3 phosphorylation in lung metastases but does not inhibit lung metastasis formation.

<p>(A-B) IHC staining of lung metastasis from mice treated with LY5. The metastatic tumor sections were stained with pSTAT3 or hematoxylin and eosin. Western blotting was performed to confirm inhibition of STAT3 phosphorylation in lung tumor sections. (C) Mice were administered OS-17 cells stably expressing luciferase by tail vein injection and subsequently treated with LY5. After 30 days, imaging was performed to evaluate the effects of LY5 on the development of pulmonary metastasis.</p