Fig. 4.

Loss of primary cilia in TRAF7-driven meningiomas. (A) Meningiomas that are either HH-dependent or -independent [based on microarray analysis, (31)] stained for Arl13b (primary cilia) and DNA (DAPI). Confocal immunofluorescence microscopy reveals that HH-dependent tumors express primary cilia, while HH-independent (TRAF7 mutated) tumors display reduced, abrogated, or absence of primary cilia. Genotypes of tumors used YNS 825: SMO L412F; YNS 422 and YNS 775: HH-driven based on gene expression analysis, mutation unknown; YNS 714: TRAF7 unknown mutation/KLF4 K409Q; YNS 670: TRAF7 K498E/KLF4 K409Q; YNS 212: TRAF7 splice-site mutation: c.1135+5G>A/ AKT E17K; YNS 560: AKT1-E17K (± TRAF7); YNS 1616: TRAF7 I368N/KLF4 K409Q; YNS 1965: TRAF7: R653Q/AKT1-E17K. 3D projections of equivalent z-stacks are shown. (Scale bar, 10 μm.) (B) Primary cultures of meningiomas that are either HH-dependent or -independent [based on microarray analysis, (31)] stained for Arl13b (primary cilia) and γ-tubulin (centrosomes, marking base of cilia). Arrowheads indicate region shown at higher magnification in inset. All images captured under identical confocal settings. [Genotypes of cultures used T633 (derived from YNS 825): SMO L412F; T343 (derived from YNS 422) and T596 (derived from YNS 775): HH driven based on gene expression analysis, mutation unknown; T554 (derived from YNS 714): TRAF7 unknown mutation/KLF4 K409Q; T524 (derived from YNS 670): TRAF7 K498E/KLF4 K409Q; T143 (derived from YNS 212): TRAF7 splice-site mutation: c.1135+5G>A/ AKT E17K); T458 (derived from YNS 560): AKT1-E17K (±TRAF7). (Scale bar: 20 μm.) (C) Schematic representation of anatomic localization of HH-dependent (turquoise) and -independent (orange) meningiomas, driver mutations, and primary cilium status. (D–E) Overexpression of V5-tagged WT and mutant (G536S or K615E) TRAF7 in HEK293 cells followed by staining for Arl13b (green), γ-tubulin (red), and V5 (white, marking TRAF7). Only mutant forms of TRAF7 affect the primary cilium. (Scale bar: 20 μm.)