Fig. 5. Loxl2 expression is modulated by Wnt and is essential for OS proliferation.

a, b qPCR analysis of collagen modifying enzymes in tumor-bearing bone (a) and primary OS cells (b) isolated from Wls^WT-OS and Wls^ΔOB-OS mice. c Loxl2 immunofluorescence (red) in Wls^ΔOB-OS and Wls^WT-OS tumors at 15 weeks. Nuclei are counterstained with DAPI. d Loxl2 immunoblot in primary tumor cells isolated from Wls^WT-OS and Wls^ΔOB-OS mice. e qPCR analysis of Loxl2 and the Wnt targets Axin2 and Alp in Fos^Tg-C3 cells stimulated with Wnt7b, Wnt9a, Wnt3a, Wnt5a or control conditioned medium (CM) for 48 h. f Loxl2 immunoblot in primary tumor cells isolated from H2-c-fosLTR OS cells treated with control or Wnt7b-CM for 48 h. g Immunoblot for Zeb1 and Zeb2 in in primary tumor cells isolated from Wls^WT-OS and Wls^ΔOB-OS mice, in Fos^Tg-C3 cells treated with Wnt7b CM and in LM7 cells ectopically expressing WNT7B. h Bone tumor burden 21 days post cell implantation in NSG mice orthotopically injected with Fos^Tg-C3 cells expressing IPTG-inducible Loxl2 shRNA or non-target shRNA and treated with IPTG or vehicle (PBS) during 18 days. Actin is used as a loading control for immunoblot. Bar graphs and plots represent or include mean ± SEM, respectively. *P < 0.05 and **P < 0.01.