Sc 11420

HT-29 cells were seeded in 100-mm dishes and treated at 50% confluency according to indicated treatment conditions, then harvested by trypsinization at 8 h after hypericin activation. Western blot analyses were conducted using total cellular protein extracts (30–50 mg). The blots were incubated overnight at +4 °C with the following specific primary antibodies: anti-HDAC1 (1/1000, ab 46985, Abcam), anti-HDAC3 (1/500, sc-11417, Santa Cruz Biotechnology), anti-HDAC6 (1/500, sc-11420, Santa Cruz Biotechnology), anti-CDKN1A (1/500, sc-397, Santa Cruz Biotechnology), anti-H3 (1:2500, ab 1791, Abcam), anti-H3ac (1:2500, MILL 17-245, Merck Millipore), followed by incubation with species-matched secondary antibodies. β-Actin (1:5000, A5441, Sigma-Aldrich) or H3 was used as the loading control. Specific proteins were detected by exposing membranes to ChemiDoc XRS+ System (Bio-Rad Laboratories) after incubation with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Densitometry analysis was performed using ImageJ software.

Puromycin-resistant, lentiviral shRNA constructs against HDAC6 or scrambled shRNA (Thermo Scientific GIPZ; Waltham, MA USA 02451) were co-transfected into Phoenix cells along with helper packaging plasmids in order to produce viruses. The jETPEI transfection reagent and protocol was used (Polyplus Transfection). Media were changed at 24 hours. Another 24 hours later, media were collected and filtered through a 0.45-μ syringe unit (BD Falcon). The breast cancer cells of interest were then transduced with the virus and selected for puromycin resistance for 48 hours and allowed to recover for another 48 hours. Protein was harvested to assess knockdown. HDAC6 antibody (rabbit polyclonal, Santa Cruz sc-11420) was used at 1:1000, for 2 hours at room temperature, and β-actin antibody (mouse, monoclonal, BD Biosciences, 558624) was used at 1:5000.