Journal: Cells

Article Title: Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia

doi: 10.3390/cells12192354

Figure Lengend Snippet: Increasing ciliary ARL13B promotes ciliary SMO enrichment in glioma cilia. ( A , B ) S7 transgenic cell lines expressing ARL13B WT :GFP ( A ) or ARL13B V358A :GFP ( B ). Cells were immunostained for acetylated alpha-tubulin (aaTUB, red) and smoothened (SMO; blue). ( C ) Zoom of boxed areas in ( A , B ). ARL13B WT :GFP + cilium (arrowhead, c1 ) colocalizes with aaTUB and SMO puncta (arrow). The ARL13B V358A :GFP + cilium is positive for aaTUB but lacks GFP (arrowhead, c2 ) and SMO puncta (arrow). ( D ) Bar graph shows % of aaTUB + cilia with SMO + cilia in untransfected (UN) (n = 182 cilia), ARL13B WT :GFP + (n = 297 cilia), and ARL13B V358A :GFP + (n = 128 cilia) S7 cells. ( E ) S7 parental cells transfected with indicated concentration of ARL13B T35N :GFP cDNA. Cells were fixed after 48 h and immunostained for SMO (red). Scale bar = 5 µm. ( F ) Quantification of results in E. Bar graph shows % of GFP + cilia with SMO + cilia after 5 (n = 26 cilia), 50 (n = 35 cilia), or 200 (n = 37 cilia) ng of ARL13B T35N :GFP cDNA/well. ( G ) Three zoomed examples from indicated cilia in ( A ) showing ARL13B WT :GFP + cilia in which the GFP (arrowhead) and SMO (arrow) signals appear to dissociate at the distal cilia tip. ( H ) Cilia of parental S7 cells treated with vehicle (PBS) ( h1 ) or recombinant human SHH [0.1 ug/µL] ( h2 – h4 ) and fixed after 24 h. SHH-treated cilia display SMO puncta (arrow) that lack or weakly express ARL13B. Scale bars in ( B ) = 10 µm, ( E , G , H ) = 5 µm. * p < 0.05, **** p < 0.0001 (ANOVA).

Article Snippet: Where indicated, indicated cell lines were treated with various drugs including cyclopamine (Calbiochem, San Diego, CA, USA; Cat #239803) (diluted in dimethyl sulfoxide (DMSO)), temozolomide (TMZ) (Sigma, St. Louis, MO, USA; Cat #T2577) (diluted in DMSO), GDC-0449 (Cayman Chemical, Ann Arbor, MI, USA; Cat #13613), SAG (Calbiochem; Cat #566661), and recombinant human SHH (high-activity) (R&D Systems, Minneapolis, MN, USA; Cat #8908-SH-005/CF), and fixed after indicated treatment durations and processed as described below.

Techniques: Transgenic Assay, Expressing, Transfection, Concentration Assay, Recombinant

Journal: Cells

Article Title: Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia

doi: 10.3390/cells12192354

Figure Lengend Snippet: ARL13B overexpression drives active SMO and GLI2 localization in glioma. ( A – D ) NIH3T3 cells were treated with vehicle (Veh), 0.1 µg/mL recombinant human SHH, 200 nM SAG, or 5 µM cyclopamine (Cyclop). After 24 h, cells were fixed and triple immunolabeled for ARL13B (green), SMO (red), and GLI2 (blue). ( E ) Quantification of the SMO fluorescence intensity within cilia of untransfected (veh) (n = 39 cilia), SHH (n = 32 cilia), and SAG (n = 44 cilia). ( F ) Quantification of the GLI2 fluorescence intensity within cilia of untransfected (veh) (n = 39 cilia), SHH (n = 32 cilia), and SAG (n = 44 cilia). ( G ) Quantification of the SMO fluorescence intensity within cilia of Veh (n = 37 cilia) or cyclop (n = 42 cilia). ( H ) Quantification of the GLI2 fluorescence intensity within cilia of Veh (n = 37 cilia) or cyclop (n = 42 cilia). ( I ) S7 parental cells immunolabeled for ARL13B (green), SMO (red), and GLI2 (blue). ( J – L ) Three examples of ARL13B WT :GFP + cilia immunolabeled for SMO and GLI2. Puncta of SMO (arrows) and GLI2 (arrowhead) are localized at the ciliary tips. ( M,N ) Bar graphs show % of endogenous ARL13B + (parental) or GFP + cilia that were SMO + ( M ) or GLI2 + ( N ). S7 parental (par) (n = 270) and ARL13B WT :GFP + (WT:GFP) (n = 70) cilia. Scale bars in ( A , I ) = 5 µm. * p < 0.05, ** p < 0.01, **** p < 0.0001 (ANOVA).

Article Snippet: Where indicated, indicated cell lines were treated with various drugs including cyclopamine (Calbiochem, San Diego, CA, USA; Cat #239803) (diluted in dimethyl sulfoxide (DMSO)), temozolomide (TMZ) (Sigma, St. Louis, MO, USA; Cat #T2577) (diluted in DMSO), GDC-0449 (Cayman Chemical, Ann Arbor, MI, USA; Cat #13613), SAG (Calbiochem; Cat #566661), and recombinant human SHH (high-activity) (R&D Systems, Minneapolis, MN, USA; Cat #8908-SH-005/CF), and fixed after indicated treatment durations and processed as described below.

Techniques: Over Expression, Recombinant, Immunolabeling, Fluorescence

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet:

Article Snippet: Recombinant Human Sonic Hedgehog Protein, High Activity , R&D , Cat# 8908-SH.

Techniques: Virus, Recombinant, Activity Assay, Protease Inhibitor, Molecular Weight, Concentration Assay, Sequencing, Modification, Affinity Purification, Expressing, shRNA, Generated, Plasmid Preparation, Software, Blocking Assay