Journal: Oncotarget

Article Title: Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

doi: 10.18632/oncotarget.6854

Figure Lengend Snippet: ( A ) Spheres of Line 0 (L0) and S3 GBM cells were dissociated, sorted as single cells into 96-well plates, and further expanded onto coverslips in 24-well plates prior to immunostaining for cilia markers after 24 hours. ( B ) Confocal maximum projection images of L0 clones (1 and 15) immunostained for PCM1 (green) and acetylated alpha-tubulin (aa-tubulin; red). The merged images (right panels) show aa-tubulin-positive axonemes (arrows) projecting from PCM1-positive basal bodies (arrowheads) in Clone 15 but not in Clone 1. ( C ) Percentage of all clones in L0 or S3 cell lines that gave rise to ciliated progeny. ( D ) Percentage of L0 or S3 cells with aa-tubulin-positive cilia derived from individual clones. Scale bar in B = 10 μm.

Article Snippet: Primary antibodies used for immunocytochemistry (ICC) or immunohistochemistry (IHC) included mouse anti-acetylated alpha-tubulin (1:3000 (ICC/IHC); Sigma (cat # T6793; lot # 088K4829)), rabbit anti-Arl13b (1:3000 (IHC); Proteintech (cat # 17711-1-AP; lot # 00017960)), mouse anti-gamma-tubulin (1:2000 (ICC/IHC); Sigma (cat # T6557; lot # 072M4808)), rabbit anti-Gli3C (1:1000 (ICC); gift from S. Scales) [ ], rabbit anti-PCM1 (1:1000 (ICC/IHC); Bethyl Laboratories (cat # A301-150A; lot # A301-150A-1)), rabbit anti-SMO (1:1000 (ICC/IHC); Abcam (cat # ab38686; lot # GR198520)).

Techniques: Immunostaining, Clone Assay, Derivative Assay

Journal: Oncotarget

Article Title: Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

doi: 10.18632/oncotarget.6854

Figure Lengend Snippet: L0, S2 and S3 cell lines were infected with lentiviral vectors encoding either mCherry alone or mCherry and dominant negative Kif3a (dnKif3a) and their cilia examined using immunocytochemistry and EM. Next, we used FAC-sorting to select mCherry-positive L0, S2, and S3 cells, expanded the sorted cells, and assessed whether they were able to form cilia using ultrastructural and immunocytochemical techniques. ( A ) Confocal image shows L0 cells (prior to sorting) with aa-tubulin-positive cilia on infected mCherry-positive cells (arrows) and non-infected mCherry-negative cells (arrowhead). ( A' ) Sorted mCherry-positive cells were fixed and analyzed by EM. The example shows a cilium with a docked basal body (arrow) and a long ciliary axoneme (arrowheads) projecting outside the cell. ( B ) L0 cells (prior to sorting) revealed aa-tubulin-positive cilia but only on non-infected (mCherry-negative) cells (arrowheads) and not on infected mCherry and dnKif3a-positive cells. ( B', B” ) By EM, sorted L0 mCherry and dnKif3a-positive cells revealed basal bodies (arrows) that lacked a clear axoneme ( B' ; arrowhead) or displayed an abnormally assembled/poorly formed axoneme ( B” ; arrowhead). ( C ) Percentage (+/− SEM) of aa-tubulin-positive ciliated cells among sorted mCherry-positive and sorted mCherry and dnKif3a-positive cells for the indicated cell lines. ( D ) Endogenous KIF3A levels are reduced in L0, S2 and S3 GBM cell lysates after dnKif3a expression. Thirty μg of total protein lysates from L0, S2 and S3 cells expressing mCherry or mCherry and dnKif3a were separated by SDS-PAGE and western blotted with an antibody against human KIF3A protein. Although KIF3A (∼ 85 kDa) was detected in all groups, lysates from mCherry and dnKif3a-expressing cells consistently displayed reduced KIF3A levels compared to those expressing mCherry alone. We also observed a smaller band (∼ 40 kDa, arrowhead) in lanes with dnKif3a-expressing cell lysates, which might either represent degraded endogenous KIF3A or possibly a sequence associated with the expression of dnKif3a. Δ-actin was run as a loading control. Scale bars for A and B = 10 μm; A' = 500 nm; B' and B” = 250 nm. *** p <0.005 (Student's t-test).

Article Snippet: Primary antibodies used for immunocytochemistry (ICC) or immunohistochemistry (IHC) included mouse anti-acetylated alpha-tubulin (1:3000 (ICC/IHC); Sigma (cat # T6793; lot # 088K4829)), rabbit anti-Arl13b (1:3000 (IHC); Proteintech (cat # 17711-1-AP; lot # 00017960)), mouse anti-gamma-tubulin (1:2000 (ICC/IHC); Sigma (cat # T6557; lot # 072M4808)), rabbit anti-Gli3C (1:1000 (ICC); gift from S. Scales) [ ], rabbit anti-PCM1 (1:1000 (ICC/IHC); Bethyl Laboratories (cat # A301-150A; lot # A301-150A-1)), rabbit anti-SMO (1:1000 (ICC/IHC); Abcam (cat # ab38686; lot # GR198520)).

Techniques: Infection, Dominant Negative Mutation, Immunocytochemistry, Expressing, SDS Page, Western Blot, Sequencing

Journal: Oncotarget

Article Title: Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

doi: 10.18632/oncotarget.6854

Figure Lengend Snippet: ( A-C ) Kaplan-Meier curves for mice xenografted with mCherry-positive control (red) or mCherry and dnKif3a-positive (blue) L0 ( A ), S2 ( B ) and S3 ( C ) cells. n = number of mice per group. ( D-F ) Sections through the mCherry-positive primary tumor mass immunostained for Arl13b, a marker of primary cilia. In L0 ( D ), S2 ( E ) and S3 ( F ) cell lines, we detected numerous Arl13b-positive cilia (arrowheads) in mCherry-positive control tumors, but not in tumors comprised of cells expressing mCherry and dnKif3a. Boxed regions in D-F are enlarged in the right panel insets to show that, in conjunction with staining for gamma-tubulin (gtub), Arl13b-positive cilia extend from gamma-tubulin-positive basal bodies (red) in mCherry control but not mCherry and dnKif3a-positive cells. Scale bars in D-F = 10 μm.

Article Snippet: Primary antibodies used for immunocytochemistry (ICC) or immunohistochemistry (IHC) included mouse anti-acetylated alpha-tubulin (1:3000 (ICC/IHC); Sigma (cat # T6793; lot # 088K4829)), rabbit anti-Arl13b (1:3000 (IHC); Proteintech (cat # 17711-1-AP; lot # 00017960)), mouse anti-gamma-tubulin (1:2000 (ICC/IHC); Sigma (cat # T6557; lot # 072M4808)), rabbit anti-Gli3C (1:1000 (ICC); gift from S. Scales) [ ], rabbit anti-PCM1 (1:1000 (ICC/IHC); Bethyl Laboratories (cat # A301-150A; lot # A301-150A-1)), rabbit anti-SMO (1:1000 (ICC/IHC); Abcam (cat # ab38686; lot # GR198520)).

Techniques: Positive Control, Marker, Expressing, Staining

Journal: Oncotarget

Article Title: Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

doi: 10.18632/oncotarget.6854

Figure Lengend Snippet: ( A ) Image of mCherry and Arl13b-positive cells (arrowheads) in satellite growths that formed outside of the primary tumor core in L0 control cell-derived tumors. ( C ) mCherry-positive cell displaying a migratory profile with a short trailing process (arrow) and long leading process (arrowheads). ( B, D ) Gamma-tubulin (gTub) (red) was substituted for mCherry in A and C, respectively. Arrows point to Arl13b-positive cilia with G-tubulin-positive basal bodies enlarged in insets. ( E ) Example of an mCherry-positive cell observed away from the primary tumor mass displaying gamma-tubulin positive basal bodies (arrowheads) and harboring a SMO-positive cilium (arrows). Scale bars for A and C = 10 μm; E = 5 μm.

Article Snippet: Primary antibodies used for immunocytochemistry (ICC) or immunohistochemistry (IHC) included mouse anti-acetylated alpha-tubulin (1:3000 (ICC/IHC); Sigma (cat # T6793; lot # 088K4829)), rabbit anti-Arl13b (1:3000 (IHC); Proteintech (cat # 17711-1-AP; lot # 00017960)), mouse anti-gamma-tubulin (1:2000 (ICC/IHC); Sigma (cat # T6557; lot # 072M4808)), rabbit anti-Gli3C (1:1000 (ICC); gift from S. Scales) [ ], rabbit anti-PCM1 (1:1000 (ICC/IHC); Bethyl Laboratories (cat # A301-150A; lot # A301-150A-1)), rabbit anti-SMO (1:1000 (ICC/IHC); Abcam (cat # ab38686; lot # GR198520)).

Techniques: Derivative Assay

Journal: MethodsX

Article Title: A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin.

doi: 10.1016/j.mex.2018.02.003

Figure Lengend Snippet: Fig. 1. Examples of differential interference contrast (DIC) and confocal fluorescence images obtained simultaneously in a Zeiss LSM 700 Axio Observer microscope and showing the same two U2OS cells live (A) and after fixation 3 min with Psuc followed by Met/Ac (B) or live (C) and after fixation 5 min with Psuc (D). (A–D) These cells stably co-expressed gTUBULIN shRNA and GFP- g-tubulin334-449, and in the images they exhibit GFP-g-tubulin334–449-labelled structures. Scale bar: 10 mm.

Article Snippet: MethodsX 5 (2018) 227–233 Contents lists available at ScienceDirect MethodsX journal homepage: www.elsevier.com/locate/mex S M 2 3 4 2 pecifications table Subject area Select one of the following subject areas: Molecular Biology More specific subject area Microscopy Method name Fixation technique Name and reference of original method https://doi.org/10.1016/j.bbamcr.2017.10.008 Resource availability http://www.sciencedirect.com/science/article/pii/S0167488917302835?via%3Dihub ethod details Live imaging followed by fixation of the imaged cells Step 1: preparation of stable cell lines Materials U2OS human osteosarcoma cells (ATCC1HTB-96TM) 35-mm tissue culture dishes Jet Pei (Polyplus Transfection, cat. no. 101-10N) pEGFP-g-tubulin334-449 [6] (Addgene Plasmid # 87859) pTER-gTUBULIN shRNA [7] (Addgene Plasmid # 87955) 35-mm MatTek glass bottom dishes (MatTek corporation, cat. no. P35G-0.170-14-C) Zeocin (ThermoFisher Scientific, cat. no. R25001) Geneticin (G418; ThermoFisher Scientific, cat. no. 10131035) Note that this list includes only necessary cell lines and non-standard laboratory equipment.

Techniques: Microscopy, Stable Transfection, shRNA