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Nocodazole

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Nocodazole is a chemical compound used in cell biology research to disrupt microtubule assembly. It functions by inhibiting the polymerization of microtubules, which are essential components of the cytoskeleton.

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Lab products found in correlation

Blebbistatin, by Cell Signaling Technology (2 mentions) Cytochalasin d, by Cayman Chemical (2 mentions) Alexa fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Rab5 c8b1, by Cell Signaling Technology (2 mentions) Anti fmnl3 ab57963, by Abcam (2 mentions) Anti galectin 3 ab2785, by Abcam (2 mentions) Anti human lamp 2 h4b4, by BioLegend (2 mentions) Anti diaph2 a300 079a t, by Fortis Life Sciences (2 mentions) Ck869, by Cell Signaling Technology (2 mentions) Dynasore, by Cell Signaling Technology (2 mentions) Gsk269962, by Cell Signaling Technology (2 mentions) Ml 141, by Cell Signaling Technology (2 mentions) Eht 1864, by Cell Signaling Technology (2 mentions) Bms 5, by Cell Signaling Technology (2 mentions) Smifh2, by Abcam (2 mentions) Ly294002, by Abcam (2 mentions) Rabbit anti n g polyclonal antibody, by Biosynth (2 mentions) Mouse anti cas9 7a9 3a3, by Cell Signaling Technology (1 mentions) Lc3a b d3u4c, by Cell Signaling Technology (1 mentions) Mg132, by Merck Group (1 mentions)

6 protocols using nocodazole

1

Characterization of Neisseria gonorrhoeae antibodies

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The purified chicken IgY anti-Neisseria gonorrhoeae antibody was raised against formalin-killed FA1090 gonococci by Cocalico Biologicals and it cross-reacts with the MS-11, FA19 and F62 strains. The rabbit anti-N.g polyclonal antibody (cat# 20-NR08) was purchased from Fitzgerald Industries. Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling—Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S). Mouse antibodies against the following antigens were purchased from Santa Cruz–Actin (sc8432), FMNL1 (sc390466), FMNL2 (sc390298), FHOD1 (sc365473), DAAM1 (sc100942). Mouse anti-Cas9 (7A9-3A3) was purchased from Cell Signaling. Anti-FMNL3 (ab57963) and anti-Galectin 3 (ab2785) mouse monoclonal antibodies were purchased from Abcam. Anti-human LAMP-2 (H4B4) was purchased from BioLegend. Anti-DIAPH2 (A300-079A-T) was purchased from Bethyl Labs. Highly cross-absorbed Alexa fluorophore conjugated secondary antibodies were purchased from Life Technologies.
Inhibitors used in this study were purchased from: (1) Cayman Chemicals—cytochalasin D (5μM), CK869 (10μM), Dynasore (30μM), GSK269962 (1μM), Nocodazole (3μM), ML-141 (5μM), EHT 1864 (5μM), BMS-5 (1μM) and (-) blebbistatin (5μM); (2) Cell Signaling—LY294002 (10μM); (3) Abcam—SMIFH2 (12.5μM to 100μM).
Ivanov S.S., Castore R., Juarez Rodriguez M.D., Circu M, & Dragoi A.M. (2021). Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages. PLoS Pathogens, 17(12), e1010184.
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2

Cell Cycle Synchronization Protocols

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Cells were synchronized in G1/S phase by double thymidine block as previously described (Moudgil et al., 2015) [43 (link)]. Cells were treated with 2 mM thymidine for 16 h with an 8 h release interval between thymidine treatments. Cell synchronization in prometaphase was performed 8 h post release from thymidine treatment by the addition of 200 ng/mL of nocodazole (Cell Signalling; 2190S) for 4 h. Cell synchronization described in Figures 5–7 (measuring NEBD to anaphase), cells were released from 2nd thymidine block for 9 h and then subjected to indicated treatments. Cell synchronization in metaphase was achieved using 25 µM MG-132 (Sigma-Aldrich; M7449).
Lewis C.W., Jin Z., Macdonald D., Wei W., Qian X.J., Choi W.S., He R., Sun X, & Chan G. (2017). Prolonged mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to paclitaxel. Oncotarget, 8(43), 73705-73722.
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3

Cytoskeleton Modulation in Cellular Injury

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Inhibitors were added in fresh growth medium 30 min prior to injury. DMSO (Fisher Scientific #BP231) was added at 0.2% (v/v) as a vehicle control. Blebbistatin (Cayman Chemical #24171) was used at 100 μM. Myosin light chain kinase inhibitory peptide 18 (P18, Cayman Chemical #19181) was used at 10 μM. ROCK inhibitor Y27632 (Cayman Chemical #10005583) was used at 10 μM. Nocodazole (Cell Signaling #2190s) was used at 100 nM. Dynasore (Sigma #D7693) was used at 60 μM. Latrunculin A (Sigma #428026) was used at 1 μM.
Griebel M., Vasan A., Chen C, & Eyckmans J. (2023). Fibroblast clearance of damaged tissue following laser ablation in engineered microtissues. APL Bioengineering, 7(1), 016112.
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4

Cell Cycle Inhibitors Protocol

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The inhibitors used in this study with their concentrations were as follows: Palblociclib (100 nM, Med Chem Express, HY-50767), CDK1i (10 μM, Sigma-aldrich, SML0569), pro-TAME (15 mM, R&D systems, I44001 M), APCin (30 μM, R&D systems, I44405 M). Nocodazole (0.1 μg/ml, Cell Signaling Technology #2190).
Awadia S., Sitto M., Ram S., Ji W., Liu Y., Damani R., Ray D., Lawrence T.S., Galban C.J., Cappell S.D, & Rehemtulla A. (2023). The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity. The Journal of Biological Chemistry, 299(6), 104786.
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5

Antibodies and Inhibitors for Gonococcal Trafficking

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The purified chicken IgY anti-Neisseria gonorrhoeae antibody was raised against formalin-killed FA1090 gonococci by Cocalico Biologicals and it cross-reacts with the MS-11, FA19 and F62 strains. The rabbit anti-N.g polyclonal antibody (cat# 20-NR08) was purchased from Fitzgerald Industries. Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling -Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S).
Mouse antibodies against the following antigens were purchased from Santa Cruz -Actin (sc8432), FMNL1 (sc390466), FMNL2 (sc390298), FHOD1 (sc365473), DAAM1 (sc100942).
Mouse anti-Cas9 (7A9-3A3) was purchased from Cell Signaling. Anti-FMNL3 (ab57963) and anti-Galectin 3 (ab2785) mouse monoclonal antibodies were purchased from Abcam. Anti-human LAMP-2 (H4B4) was purchased from BioLegend. Anti-DIAPH2 (A300-079A-T) was purchased from Bethyl Labs. Highly cross-absorbed Alexa fluorophore conjugated secondary antibodies were purchased from Life Technologies.
Inhibitors used in this study were purchased from: (1) Cayman Chemicals -cytochalasin D (5µM), CK869 (10µM), Dynasore (30µM), GSK269962 (1µM), Nocodazole (3µM), ML-141 (5µM), EHT 1864 (5µM), BMS-5 (1µM) and (-) blebbistatin (5µM); (2) Cell Signaling -LY294002 (10µM); (3) Abcam -SMIFH2 (25µM).
Ivanov S.S., Castore R., Circu M.L., & Dragoi A. (2021). Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages.
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6

Cytoskeleton Dynamics in Hypo-osmotic Stress

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Vimentin-EGFP, vimentin-mCherry, nestin-GFP, and tubulin-GFP were kind gifts from John Eriksson (Turku, Finland). The hypo-osmotic medium was made by using complete growth medium diluted appropriately in deionized water (dilution 1:9 to obtain 30 mOsm, 1:4 to obtain 60 mOsm, and 1:1 to obtain 150 mOsm) and used in all the experiments except Figure S1C. Alternatively, dialyzed serum was added into diluted DMEM with deionized water in Figure S1C which was used to verify the phenotype. Microtubule assembly was inhibited by 10 μM nocodazole for 1.5 hours (#2190S, Cell Signaling, Danvers, MA). Dynein was inhibited by 20 μM ciliobrevin D for 45 minutes (#M60041; Xcessbio, San Diego, CA) under serum deprivation to facilitate its efficient inhibition. Actin assembly was inhibited by 5 μg/mL cytochalasin D for 1 hour (#PHZ1063; Thermo Fisher Scientific, Waltham, MA).
Li J., Gao W., Zhang Y., Cheng F., Eriksson J.E., Etienne-Manneville S, & Jiu Y. (2019). Engagement of vimentin intermediate filaments in hypotonic stress. Journal of cellular biochemistry, 120(8).
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