Stealth sirna odf2mss207236

Plasmid DNA or siRNA was transfected using EndoFectin™ Max Transfection Reagent following the manufacturer´s instructions (GeneCopoeia #EF014). The mouse Foxa1 gene (NM_008259) tagged with Myc-DDK in pCMV6-Entry was obtained from Origene (MR225487). The coding region of Foxa1 was cloned in-frame to Egfp in pEGFP-N1 (Clontech).
Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).
For Odf2 knockdown, Odf2 siRNA (stealth siRNA ODF2MSS207236; Life Technologies; final concentration 40 nM) and control siRNA (siGenome Non-targeting siRNA #1; ThermoFisher Scientific Biosciences) were used. Additionally, the short hairpin constructs sh3 (specifically targeting sequence gaactcctccaggagatac of mouse Odf2/Cenexin;^{50 (link)}) or K07 (Origene), which functions as control while lacking homology with any known mRNA, were used. For rescue, the expression plasmid encoding human Cenexin (hCenexin)^{46 (link)}) was co-transfected, and to identify transfected cells, human histone H4^{84 (link)} fused to egfp was also co-transfected.

NIH3T3 cells were seeded at a density of 1.25 × 10⁵ on coverslips in 6-well plates. Transfection was conducted 24 h post-seeding, and cells were processed for immune-cytology 24 h later. Plasmid DNA or siRNA were transfected using EndoFectin^TM Max Transfection Reagent, following the manufacturer’s instructions (#EF014, GeneCopoeia, Inc., Rockville, MD, USA). For Odf2 knockdown, the short hairpin constructs sh3 (specifically targeting sequence gaactcctccaggagatac of mouse Odf2/cenexin; [61 (link)] or Odf2 siRNA (stealth siRNA ODF2MSS207236; final concentration 40 nM; Life Technologies, Carlsbad, CA, USA) were used. As controls, the plasmid K07 (OriGene Technologies, Inc., Rockville, MD, USA), which lacked homology with any known mRNA, or the control siRNA (siGenome Non-targeting siRNA #1; D-001210-01-05; target sequence UAGCGACUAAACACAUCAA; Dharmacon, Lafayette, CO, USA) were used. Additionally, for rescue, the expression plasmid encoding human cenexin (hCenexin) [62 (link)] was co-transfected. The co-transfection of pCentrin-2::Cherry was used to identify the centrosome in the transfected cells.