RPE-1, HCT 293 and HCT 116 cells were cultured as described previously (Kiyomitsu and Cheeseman, 2012 (link)). Cells were treated with DMSO, 50 μM palmostatin, 10 μM Wnt-C59, or 5 μM Qz for 12 hours. For live-cell imaging, cells were cultured with 50 ng/ml Hoechst33342 for 10 minutes prior to observation. RNAi experiments were carried out with 27mer siRNA duplexes corresponding to regions of PORCN and LYPLA1. The oligonucleotides (Origene) were transfected at an amount of 100 pmol per 6 well plate into HCT 293 cells using the Lipofectamine 2000 system (Thermo Fisher) in accordance with the supplier’s recommendations. Cells were fixed with methanol for 3 minutes followed by 3 washes in PBS 0.3% triton X 100 for DNA (Hoeschst3342) and microtubule (1:2000 E7 anti-beta tubulin antibody) staining. For importin α staining (1:1000 antibody gift from Karsten Weis), cells were fixed with 3% paraformaldehyde and 2% sucrose. Images were acquired on an Andor/Nikon spinning disk confocal. For both fixed and live cells, 10 Z sections were acquired in 1 μm steps using a Nikon Plan-fluor 40x/0.75 NA objective. Distance measurements and mean fluorescent intensities were analyzed using ImageJ/Fiji software.
Oligonucleotides
Manufactured by OriGene
Sourced in United States
Oligonucleotides are short, synthetic DNA or RNA molecules that can be used as probes, primers, or therapeutic agents in various laboratory and research applications. They are designed to be complementary to specific target sequences, allowing for targeted analysis, detection, or manipulation of genetic material.
Automatically generated - may contain errors
2 protocols using oligonucleotides
1
Microscopic Analysis of Cellular Response
2
CRISPR-Mediated TDP-43 Knockdown in U-2 OS Cells
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Oligonucleotides targeting TDP-43 were ordered from Integrated DNA Technologies (Integrated DNA Technologies, Coralville, IA, USA). Oligonucleotides were annealed and cloned into pCas-Guide (Origene, Rockville, MD, USA) according to manufacturer’s protocol. gRNA targeted the following sequence: GTTTGTGGGGCGCTGTACAG. Cloned pCas-Guide was co-transfected with pDonor-D09 (GeneCopoeia, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) into U-2 OS cells. The following day, cells were treated with puromycin (1.5 μg/mL). After 48 h of selection, puromycin was removed. Single-cell clones were generated by limiting dilution.
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