Figure 6
Figure 6. L-4F has anti-myeloma effects in vivo. (A) Western blot of the different molecular weight isoforms of adiponectin in serum of KaLwRij mice treated with L-4F for 28 days. Each lane represents serum from one mouse. (B) KalwRij mice were treated with L-4F for 28 days before inoculation of 5TGM1 myeloma cells. Treatment was continued for a further 25 days, at which point mice were killed. The proportion of GFP-positive myeloma cells in BM was quantitated by flow cytometry. **P < .01, compared with vehicle control. (C) KaLwRij mice were treated as described in panel B and tumor burden monitored by serum IgG2bκ ELISA. Two-way ANOVA demonstrated a significant difference in the rate of tumor development in L-4F-treated mice compared with control (P < .01). Days 0 to 15 are expanded in the second panel to demonstrate differences in tumor burden detectable at early time points. (D) Apoptotic myeloma cells in the BM were quantitated by TUNEL staining. A representative image is shown. White asterisks represent TUNEL-positive cells. Bar represents 50 μm. *P < .05, compared with vehicle control. (E) Proliferation was quantitated in the BM by immunostaining for Ki-67. Bars represent 200 μm. *P < .05, compared with vehicle control (Vehicle, n = 9; L-4F, n = 10). (F) Adiponectin-deficient mice (KO) or WT controls were treated with L-4F for 28 days before inoculation of 5TGM1 myeloma cells. Treatment was continued for a further 25 days, at which point mice were killed. Tumor burden was monitored by serum IgG2bκ ELISA. Data are expressed as percentage change in serum IgG2bk after treatment with L-4F. #P < .05 compared with WT. (G) KaLwRij mice were treated with L-4F for 28 days before inoculation of 5TGM1 myeloma cells. Treatment was continued until time of paraplegia, as a surrogate for survival. Data are displayed as a Kaplan-Meier plot, and a log-rank (Mantel-Cox) test demonstrated a significant increase in survival with L-4F treatment (P < .0001, n = 15). (H) KaLwRij mice were inoculated subcutaneously with 5TGM1 myeloma cells and treated with L-4F. Tumor volume was measured daily (Vehicle, n = 7; L-4F, n = 8). Two-way ANOVA demonstrated a significant difference in the rate of tumor development in L-4F-treated mice compared with control (P < .05). Data are mean ± SEM.

L-4F has anti-myeloma effects in vivo. (A) Western blot of the different molecular weight isoforms of adiponectin in serum of KaLwRij mice treated with L-4F for 28 days. Each lane represents serum from one mouse. (B) KalwRij mice were treated with L-4F for 28 days before inoculation of 5TGM1 myeloma cells. Treatment was continued for a further 25 days, at which point mice were killed. The proportion of GFP-positive myeloma cells in BM was quantitated by flow cytometry. **P < .01, compared with vehicle control. (C) KaLwRij mice were treated as described in panel B and tumor burden monitored by serum IgG2bκ ELISA. Two-way ANOVA demonstrated a significant difference in the rate of tumor development in L-4F-treated mice compared with control (P < .01). Days 0 to 15 are expanded in the second panel to demonstrate differences in tumor burden detectable at early time points. (D) Apoptotic myeloma cells in the BM were quantitated by TUNEL staining. A representative image is shown. White asterisks represent TUNEL-positive cells. Bar represents 50 μm. *P < .05, compared with vehicle control. (E) Proliferation was quantitated in the BM by immunostaining for Ki-67. Bars represent 200 μm. *P < .05, compared with vehicle control (Vehicle, n = 9; L-4F, n = 10). (F) Adiponectin-deficient mice (KO) or WT controls were treated with L-4F for 28 days before inoculation of 5TGM1 myeloma cells. Treatment was continued for a further 25 days, at which point mice were killed. Tumor burden was monitored by serum IgG2bκ ELISA. Data are expressed as percentage change in serum IgG2bk after treatment with L-4F. #P < .05 compared with WT. (G) KaLwRij mice were treated with L-4F for 28 days before inoculation of 5TGM1 myeloma cells. Treatment was continued until time of paraplegia, as a surrogate for survival. Data are displayed as a Kaplan-Meier plot, and a log-rank (Mantel-Cox) test demonstrated a significant increase in survival with L-4F treatment (P < .0001, n = 15). (H) KaLwRij mice were inoculated subcutaneously with 5TGM1 myeloma cells and treated with L-4F. Tumor volume was measured daily (Vehicle, n = 7; L-4F, n = 8). Two-way ANOVA demonstrated a significant difference in the rate of tumor development in L-4F-treated mice compared with control (P < .05). Data are mean ± SEM.

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