Primary HESCs at 80% confluency were transfected with DNA vectors or small interfering RNA (siRNA) oligonucleotides by the calcium phosphate co-precipitation method using the ProFection mammalian transfection kit (Promega, Madison, WI) according to the manufacturer's instructions. Reporter assays were done in 96-well plates. The X-box binding protein 1 (pcDNA3/XBP1-luc) reporter construct was a kind gift from Dr. Etsu Tashiro (Keio University, Tokyo, Japan). A constitutively active renilla expression vector (pRL-sv40) served as an internal transfection control. The plates were washed twice in phosphate-buffered saline (PBS) and firefly and Renilla activities were measured using the Luclite luciferase reporter assay system (Luclite, PerkinElmer, Boston, MA) and the luminescence was measured on a Victor II plate reader (PerkinElmer). For gene-silencing studies, HESCs were cultured in 6-well plates until 80% confluency and transiently transfected with 100 nM of the following siRNA reagents (Dharmacon, Lafayette, CO): siCONTROL non-targeting (NT) siRNA pool and HSPA8 siGENOME SMARTpool. All experiments were performed on three or more primary cultures from different endometrial biopsies.
Nt sirna pool
Manufactured by Horizon Discovery
The NT) siRNA pool is a collection of small interfering RNAs (siRNAs) designed to target multiple genes simultaneously. It is a versatile tool that can be used for gene knockdown experiments in cell-based studies. The pool contains a mixture of individual siRNA duplexes that work together to achieve effective gene silencing, providing a convenient and efficient approach for exploring the effects of simultaneous knockdown of multiple genes.
Automatically generated - may contain errors
2 protocols using nt sirna pool
1
Transfection and Reporter Assays in Primary HESCs
2
EMT Markers Expression Analysis
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Coding sequence of Snail, Slug, Zeb1 and Twist were amplified from MGC cDNA clones (Thermo Fisher) and cloned into pcDNA 4/TO vector. E-Cadherin and Vimentin coding sequences were amplified from Huh7.5 or Huh7.5M cDNAs and cloned into pcDNA 4/TO. All the clones were confirmed by sequencing. PL-SIN-Nanog-EGFP was from Addgene49 (link)(plasmid 21321). For ectopic over-expression, cells were transfected with pcDNA4/TO empty vectors or those expressing the desired protein. 48 hrs post transfection, expression of EMT markers was analyzed by immunobloting. Cells stably expressing any of these proteins were generated by selection under Zeocin. For Snail depletion, the cells were transfected with specific or non-targeting (NT) siRNA pool (Dharmacon). For Slug depletion, Huh7.5M cells were transfected with NT shRNA vector pools or those targeting Slug (Thermo Fisher). In both cases, cells were lysed at 72 hrs post transfection and processed for immunobloting.
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