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. 2020 Jul 20;8:114. doi: 10.1186/s40478-020-00994-7

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — Primary meningioma cells accumulate Smoothened in cilia, but do not transduce ciliary Hedgehog signals. a, b Confocal immunofluorescence microscopy for the ciliary markers acetylated tubulin (AcTub) or Arl13b, the ciliary base/centriole marker Centriolin, and DNA (Hoechst) reveals that M10G meningioma cells express primary cilia, but BenMen meningioma cells do not, instead expressing AcTub in a perinuclear pattern. c Confocal immunofluorescence for SMO 72 h after transfection of SMO, oncogenic point substitutions of SMO (SMO^W535L or SMO^L412F), or empty vector (EV) control, shows that exogenous SMO accumulates in the cilia of M10G meningioma cells. d qRT-PCR assessment of GLI1 expression 72 h after transfection of SMO, SMO^W535L, SMO^L412F, constitutively active GLI2 (GLI2^CLEG), or EV control, shows that over-expression of SMO activates the Hedgehog transcriptional program in NIH/3T3 cells, but not in BenMen or M10G meningioma cells. In contrast, over-expression of GLI2^CLEG mildly induces GLI1 expression in meningioma cells (*p < 0.01 Student’s t test). e qRT-PCR assessment of GLI1 expression 24 h after treatment with recombinant Sonic Hedgehog (SHH, 1 μg/ml), SMO agonist (SAG, 100 nM), or vehicle control, demonstrates that pharmacologic stimulation of the Hedgehog pathway activates the Hedgehog transcriptional program in NIH/3T3 cells, but not in BenMen or M10G meningioma cells (*p < 0.0001 Student’s t test). f qRT-PCR assessment of GLI1 expression shows that CRISPRi suppression of PTCH1 (sgPTCH1) compared to transduction of non-targeted sgRNAs (sgNTC) fails to activate the Hedgehog transcriptional program in M10G meningioma cells (*p < 0.0001 Student’s t test). g qRT-PCR assessment of GLI1 expression shows that CRISPRi suppression of SMO (sgSMO) compared to transduction of sgNTC fails to suppress the Hedgehog transcriptional program in M10G meningioma cells (p < 0.0001 Student’s t test). h Quantitative confocal immunofluorescence microscopy for the proliferation marker Ki67 demonstrates that CRISPRi suppression of PTCH1 or SMO compared to sgNTC fails to alter M10G meningioma cell proliferation. qRT-PCR data are normalized to GAPDH expression and EV, vehicle, or sgNTC controls as indicated. All experiments were performed at least 3 times with at least 3 technical replicates