Mouse anti histone h3

Standard IF procedures were used. Briefly, cells were permeabilized (0.5 % Triton X-100 in PBS) for 15 min and incubated with blocking solution (DAKO #X0909) for 30 min. The following primary antibodies were used: goat anti-V5 (Bethyl #A190-119A) 1:200; mouse anti-FLAG (Sigma #F1804) 1:200; rabbit anti-BRD4 (Abcam #ab128874) 1:200; rabbit anti-TRIM24 (Proteintech #14208-1-AP) 1:400; rabbit anti-K3K23Ac (Cell Signaling #8848) 1:200, and mouse anti-histone H3 (Active Motif #39763) 1:4000. Alexa Fluor-conjugated secondary antibodies were from Invitrogen and Hoechst 33342 (Invitrogen, #H3570) was used for nuclei counterstaining (10 µg/ml). For each Alexa fluorochrome, images (4–10 fields per well; 20X magnifications) were acquired using the Operetta High-Content Screening System (PerkinElmer). Image analysis was performed in Harmony software (PerkinElmer) by selecting nuclei and cytoplasm in Hoechst channel. Gating for single cells of flat morphology was done based on nuclei area, roundness and intensity of Hoechst in the nucleus and in the cytoplasm. In every gated cell, the mean signal intensity value for each fluorochrome was calculated separately in nucleus and cytoplasm. The specific nuclear signal was estimated subtracting mean cytoplasmic value from nuclear; and average value was calculated using at least 1000 cells in each well.

The following primary antibodies (at the dilution indicated) were used for either immunofluorescence or Western blotting: rabbit anti-Ki67 (1:100, cod. HPA001164; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-p21 (1:100, cod. B1313; Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-Histone H3 (1:500, cod. 61475; Active motif, Carlsbad, San Diego, CA, USA); mouse anti-LC3 (1:100, cod. NB600-1384; Novus Biologicals, Milano, Italy); rabbit anti-LC3 (1:1000, cod. L7543; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-LAMP1 (1:1000, cod. 555798; BD, Biosciences, Franklin Lakes, NJ, USA); rabbit anti-β-Catenin (1:500, cod. PA5-77934; Invitrogen, Paisley, UK); rabbit-anti ATG7 (1:500, cod. AB10511; Millipore, Burlington, MA, USA); mouse-anti ATG7 (1:500, cod. SAB4200304; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-β-tubulin (1:1000, cod. T5326; Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-GAPDH (1:1000, cod. G9545; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-β-actin (1:2000, cod. A5441; Sigma-Aldrich, St. Louis, MO, USA).

The following primary antibodies were employed for Western blotting: mouse anti-β-tubulin (1:1000, cod. T5201; Sigma-Aldrich Corp.), mouse anti-β-actin (1:2000, cod. A5441; Sigma-Aldrich Corp.), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma-Aldrich Corp.), mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem, St. Louis, MO, USA), mouse anti-histone H3 (1:500, cod. 61475; Active Motif, Carlsbad, CA, USA). The secondary antibodies used for Western blot analysis were the following: horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000, cod. 170-6516; Bio-Rad, Hercules, CA, USA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, cod. 170-6515: Bio-Rad, Hercules, CA, USA). The primary antibodies employed for immunofluorescence staining are listed below: mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem), rabbit anti-cathepsin D (1:100; EMD Biosciences, Calbiochem, San Diego, CA, USA), rabbit anti-p27 (1:100, cod. 2552; Cell Signaling, Danvers, MA, USA), rabbit anti-Ki-67 (1:100, cod. HPA001164; Sigma-Aldrich), mouse anti-E-cadherin (1:50, cod. 14472S; Cell Signaling) and rabbit anti-N-cadherin (1:50, cod. 4061S; Cell Signaling). The secondary antibodies were goat-anti rabbit IgG Alexa Fluor Plus 488 (1:1000, cod. A32731; Invitrogen, Waltham, MA, USA) and goat-anti mouse IgG Alexa Fluor Plus 555 (1:1000, cod. A32727; Invitrogen).