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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Nat Cell Biol. 2015 Jan 12;17(2):170–182. doi: 10.1038/ncb3090

Figure 2. Disrupting POSTN by shRNA markedly inhibited tumor growth and reduced TAM density in GSC-derived xenografts.

Figure 2

a, Immunoblot analysis of POSTN in GSCs expressing non-targeting shRNA (shNT) or POSTN shRNA (shPOSTN). Targeting POSTN by two distinct shRNA clones O56 and O57 through lentiviral infection reduced POSTN expression by 85–90% in GSCs (T387). b, In vivo bioluminescent imaging analysis of tumor growth in mice bearing GBM xenografts derived from GSCs expressing shNT or shPOSTN. GSCs (T387) were transduced with firefly luciferase and shPOSTN or shNT and then engrafted intracranially into athymic nude mice. Representative images on day 15 post transplantation were shown (data from 5 mice). Silencing POSTN by both O56 and O57 shRNA clones dramatically delayed tumor growth. c, Kaplan-Meier survival curves of mice implanted with GSCs expressing shPOSTN or shNT (control). GSCs were transduced with shPOSTN (O56 or O57) or shNT through lentiviral infection and then were transplanted intracranially into athymic nude mice (20,000 cells per mouse). Survival curves were analyzed by two-tailed log-rank test. Mice bearing GSC-derived tumors expressing shPOSTN showed a significant survival extension relative to the control group. ***, p<0.001 (n=6 mice for shNT and O57, and n=8 mice for O56). d, Immunofluorescent analysis of POSTN (red) and the TAM marker Iba1 (green) in GBM tumors derived from GSCs expressing shPOSTN or shNT. Frozen sections of GBM tumors derived from GSCs with shPOSTN (O56 and O57) or shNT were immunostained with antibodies against POSTN and Iba1 and counterstained with DAPI (blue). Marked reductions of POSTN and Iba1 (TAMs) signals were detected in the GSC-derived tumors expressing shPOSTN. Scale bar, 80μm. e and f, Graphical analyses of (d) showing a decrease of POSTN signal intensity by ~80% and a significant reduction of TAM density by ~60% in the GSC-derived tumors expressing shPOSTN. POSTN intensity and TAM density were analyzed with ImageJ. *, p<0.05; ***, p<0.001 (n=5 tumors; mean ± s.e.m.; two tailed unpaired t-test).