Cells were plated onto Matrigel-coated coverslips and serum starved for 24 h as described above. Ciliobrevin (30 µM) (Sigma-Aldrich; 250401-10MG) was added to the cultures for 1 h at 37°C before fixing cells with 4% PFA for 15 min at 37°C. Samples were processed as described above, except that PBST was used for washes to remove autofluorescent residue left by the drug.
Ciliobrevin
Manufactured by Merck Group
Ciliobrevin is a laboratory reagent used for research purposes. It is a small molecule inhibitor that targets the enzyme dynein, which is involved in the movement of cilia and flagella. Ciliobrevin is utilized in experimental settings to investigate the role of dynein in cellular processes.
Automatically generated - may contain errors
3 protocols using ciliobrevin
1
Ciliobrevin Treatment of Cells
2
HUVEC Cell Culture and Immunofluorescence
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Cells were seeded at 8 × 104 cells/ml into Ibidi µ-slide eight-well plates. HUVECs were grown for 72 h. Cells were starved in serum-free M199 plus 10 mM Hepes medium for 1 h before treatments. Histamine was used at 30 µM unless otherwise stated, epinephrine hydrochloride (Sigma-Aldrich; E4642) was used at 100 µM in solution with 100 µM IBMX (Sigma-Aldrich; I5879), and thrombin (Sigma-Aldrich; T1063) was used at 2.5 U/ml. Cells were pretreated with 1 µM nocodazole (Sigma-Aldrich; SML1665), 40 µM ciliobrevin (Sigma-Aldrich; 25401), 10 µM AM-BAPTA (Tocris; 2787), and 10 µM H89 (Tocris; 2910/1) in related experiments before histamine stimulation. Cells were fixed with 4% PFA for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 solution. Cells were incubated with primary antibody (in PBS; see Table S2 for primary antibodies) for 1 h followed by fluorescently labeled appropriate species secondary antibodies for 30 min (see Table S3 for secondary antibodies). Cells were briefly incubated in Hoechst before being mounted with Ibidi mounting medium.
3
Microtubule Dynamics in Zebrafish Ovaries
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Ovaries we re dissected from Tg(buc:buc-gfp-buc 3'utr) or Tg(bact:EMTB-3XGFP) juvenile fish (6-8 wpf, SL ~10-15mm) into fresh warm HL-15 media as described in (Elkouby & Mullins, 2017b) .
Then HL-15 was replaced with HL-15 containing either 20μM nocodazole (Abcam #ab120630), 50μM colchicine (Sigma Aldrich #C9754), 25μM ciliobrevin (Sigma Aldrich #250401), 40μM taxol (Abcam #ab120143) or an equivalent volume of DMSO. Each drug treatment experiment contained DMSO groups and Tg(bact:EMTB-3XGFP) as controls. Ovaries we re incubated for 90min at 28°C in the dark.
Ovaries we re then mounted for live imaging in media containing their respective drug concentration or DMSO control. Ovaries we re still healthy and viable for at least 8hrs after the 90min time point, verified by 500nM MitoTracker Mitochondria-Selective Probes (Molecular Probes #M7512). DMSO-treated ovaries always showe d normal intact microtubules.
Then HL-15 was replaced with HL-15 containing either 20μM nocodazole (Abcam #ab120630), 50μM colchicine (Sigma Aldrich #C9754), 25μM ciliobrevin (Sigma Aldrich #250401), 40μM taxol (Abcam #ab120143) or an equivalent volume of DMSO. Each drug treatment experiment contained DMSO groups and Tg(bact:EMTB-3XGFP) as controls. Ovaries we re incubated for 90min at 28°C in the dark.
Ovaries we re then mounted for live imaging in media containing their respective drug concentration or DMSO control. Ovaries we re still healthy and viable for at least 8hrs after the 90min time point, verified by 500nM MitoTracker Mitochondria-Selective Probes (Molecular Probes #M7512). DMSO-treated ovaries always showe d normal intact microtubules.
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