Supplementary Figure 3: Silencing POSTN by shRNA reduced TAM density and extended survival of mice bearing the GSC-derived xenografts. | Nature Cell Biology

Supplementary Figure 3: Silencing POSTN by shRNA reduced TAM density and extended survival of mice bearing the GSC-derived xenografts.

From: Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth

Supplementary Figure 3

(a) Immunoblot analysis of POSTN in GSCs expressing non-targeting shRNA (shNT) or POSTN shRNA (shPOSTN, O55). Disrupting POSTN by the shRNA clone O55 through lentiviral infection reduced POSTN expression by >90% in GSCs. (b,c) Kaplan–Meier survival curves of mice implanted with GSCs expressing shPOSTN or shNT (control). GSCs (T3691 or T4121) were transduced with shPOSTN (O55, O56, or O57) or shNT through lentiviral infection and then transplanted intracranially into athymic/nude mice (20,000 cells per mouse). Mice bearing GSC-derived tumours expressing shPOSTN showed a significant survival extension relative to the control group. (n = 5 mice for each group; P = 0.0477 for T3691-O55 versus shNT; P = 0.0026 for T4121-O56 versus shNT; P = 0.0027 for T4121-O57 versus shNT; two-tailed log-rank test). (d) Immunofluorescent analysis of the TAM marker Iba1 (green) in GBM tumours derived from GSCs expressing shPOSTN (O55) or shNT. Frozen sections of GBM tumours derived from GSCs (T3691) expressing shPOSTN (O55) or shNT (NT) were immunostained with antibodies against Iba1 and counterstained with DAPI (blue). A dramatic reduction of TAMs was detected in the GSC-derived tumours expressing shPOSTN. Scale bar, 40 μm. (e) Graphical analysis of (d) showing a significant reduction of TAM density by ∼70% in the GSC-derived tumours expressing shPOSTN. POSTN intensity and TAM density were analysed with ImageJ. ∗∗, P < 0.01 (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test).

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