Analysis of STAT phosphorylation in spleens after 21 days of treatment. (A) Immunohistochemistry with antibodies against pSTAT5 of spleen sections from mice that were killed 2 hours after the final dose. Scale bars indicate 100 μm. (B) Quantification of the pSTAT5 immunohistochemistry with Definiens image analysis software. The histogram shows the percentage of pSTAT5⁺ nuclei (mean ± SEM of 4-8 mice per group). Note that the phosphorylation of STAT5 in the HU-treated group was significantly elevated compared with the vehicle-treated group, whereas the pSTAT5 in the INC424-treated groups was lower. (C) Levels of phosphorylated and total STAT3 and STAT5 proteins in spleen homogenates were assessed by immunoblot analysis. (D) Relative levels of pSTAT5 and total STAT5 were measured in spleen homogenates using MSD technology. The values represent the mean ± SEM, n = 8 per group. (E) Measurement of EPO levels in spleen samples after therapy. Spleen samples were homogenized and levels of mouse EPO were determined by ELISA. Values represent means ± SEM (n = 7-8). To fit the results on the graph, the HU-treated samples are shown in a 10-fold expanded scale. *Significantly different from vehicle-treated control group using Mann-Whitney rank sum test (P < .05).