Figure 3 from Inhibition of Extracellular Matrix Protein Fibulin-3 Reduces Immunosuppressive Signaling and Increases Macrophage Activation in Glioblastoma

Fibulin-3 regulates the expression of immunosuppressive signals in GBM cells. A, Transient transfection of fibulin-3 siRNAs in GSCs results in decreased expression of CSF-1, TGF-β1, and the innate immune checkpoint CD47. B, Same results as in (A), validated by Western blotting. Total protein loading (amido black stain of blotting membranes) was used as a loading control to normalize the expression of proteins detected in culture medium. The numbers indicate relative integrated optical density for each band, normalized to the loading control and to total protein measurement. ctrl, control; fib-3, fibulin-3. C, qRT-PCR of human fibulin-3 (FBLN3) and immunosuppressive signals detected in intracranial GSC-derived tumors expressing control or fibulin-3 short hairpin RNAs (shRNA; human-specific primers were used to detect human mRNA in tissue resected from mouse brains). Statistical analysis performed by multiple paired t tests with multiple comparison correction. D, Expression of CSF-1 and CD47 in the GBM cell lines U251MG and GL261, transfected to overexpress fibulin-3. Statistical analysis by one-way ANOVA for each cell line.