Silencing Kindlin-3 in HL-60 Cell Differentiation

HL-60 cells were maintained in Advanced RPMI containing 10% FBS and 1% pen/strep. HL-60 cells were washed once a day for a minimum of three days to ensure optimal growth. These HL-60s were transfected with control scrambled siRNA or kindlin-3-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by electroporation using the Amaxa Nucleofector 4D using the SF Cell line 4D-nucleofector X Kit L (Lonza, Basel, Switzerland), according to the manufacturer’s instructions and as described previously [4 (link),5 (link)]. HL-60 cells were given fresh media 24 h before electroporation to maximize survival. Cells were pelleted from the media, resuspended in high-resistance nucleofection buffer in the presence of 100 nM siRNA, and electroporated. Immediately after electroporation, cells were transferred to 37 °C media containing 1.3% DMSO and differentiated for 3–5 days under the influence of siRNA prior to experimentation [4 (link),5 (link)]. It was confirmed by Western blot that kindlin-3 expression was silenced up to 90% in dHL-60 cells. Kindlin-3 siRNA-scrambled mutation R556A was used as a control.