Sc 29350 sh

For cell line transfection, SH-SY5Y cells were seeded on 6-well plates at a density of 6 × 10⁵ cells/plate and cultured overnight. The SH-SY5Y cells were transfected with Hsp27 shRNA plasmid (sc-29350-SH, Santa Cruz Biotechnology) or control shRNA plasmid (sc-108060, Santa Cruz Biotechnology) using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s protocol when at ~80% confluence for 72 hour. For adenoviral transient infection, primary neurons were infected with the recombinant adenovirus carrying scramble RNA (Ad-scramble), short hairpin RNA-Hsp27 (Ad-shHsp27), empty vector (Ad-vector) or Hsp27 expression plasmid (Ad-Hsp27) at a multiplicity of infection of 100 for the indicated hours. The knockdown efficiency was determined by immunofluorescence staining or western botting.

The human non–small lung cancer cell line NCI-H460 was cultured in RPMI (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco) in a 37°C incubator with 5% CO₂. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA USA). Lentiviruses were used to create stable NCI-H460 cell lines expressing shRNA for HSP27 (puromycin resistance gene). The HSP27 shRNA plasmid (sc-29350-SH), and shRNA plasmid transfection reagent (sc-108061) were ordered from Santa Cruz Biotechnology. To generate the sh-Control, and sh-HSP27 cells, the cell lines were transduced with 1 mol of lentivirus and selected using puromycin (1 ug/mL) for at least one week.