Fig 7. PKHB1 reduced in vivo CLL tumor burden by inducing PLCγ1 activation and PCD.

(A) NSG mice were subcutaneously transplanted with MEC-1 cells. Starting 14 d after the engraftment, the mice received a weekly intraperitoneal injection of vehicle (control), PKHB1 (left panel), or 4N1K (right panel). Tumor volume was measured using a caliper and graphed. The data are presented as mean ± SD (n = 8). In contrast to vehicle or 4N1K treatment, PKHB1 treatment significantly reduced the tumor volume. (B) In an experiment similar to (A), tumor growth was visualized by measuring glucose uptake. The color scale indicates the fluorescence intensity. (C) The hemoglobin (Hb) concentration (n = 8 per group) was assessed in vehicle- (control) or PKHB1-treated tumors. The bars indicate the mean of the data obtained. (D) Tumor vascularity was investigated by immunohistochemistry analysis of mCD31. Representative photographs and visual quantification of microvessel density (right panel) confirmed similar vascularity in tumors from vehicle- and PKHB1-treated mice. The data are presented as mean ± SD. (E) PLCγ1-Y783 phosphorylation was assessed in tumors obtained from vehicle- (control) and PKHB1-treated mice. Phospho-PLCγ1 was quantified by flow cytometry using MFI. The data are presented as mean ± SD (n = 6). (F) Calreticulin exposure was assessed in tumors from vehicle- (control) and PKHB1-treated mice and graphed. The data are presented as mean ± SD (n = 6). (G) Percentage of cell death was measured by Annexin-V-positive/PI-positive staining in tumors obtained from vehicle- (control) and PKHB1-treated mice. The data are plotted as mean ± SD (n = 8). The statistical analyses included in this figure were performed with the Mann-Whitney test. A.U., arbitrary units; NS, not significant.