Anti histone h3

The protein profiles of M, MP, and MP@H-MnO₂-Dox-Col NP were evaluated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie brilliant blue staining (Beyotime, Shanghai, China). Western blotting was conducted to identify specific protein markers in RAW264.7 cells. After transferring the proteins onto polyvinylidene difluoride membranes, these were incubated at 4℃ overnight with anti-histone H3 (BioLegend, San Diego, CA, USA), anti-pan-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA), and integrin α4 antibodies (Proteintech, Chicago, USA) and Na⁺-K⁺-ATPase (Abcam, Cambridge, UK), which was used as a reference protein.

Extracts were prepared using a Cytoplasmic and Nuclear Extraction kit (Nordic BioSite) following the manufacturer's instructions. Briefly, cells were washed with ice cold PBS and the pellet was resuspended into the volume equal to seven times of cytoplasmic extraction reagent. After 30 min incubation on ice, the lysate was centrifuged at 16000 x g for 10 min at 4°C. Supernatant (cytoplasmic fraction) was carefully removed and the pellet was resuspended into the volume equal to two times of the nuclear extraction buffer. Lysates were placed on ice for 30 min with occasional vortexing followed by centrifugation at 16000 x g for 10 min at 4°C. Supernatant (nuclear fraction) was collected. Immunoblotting was performed using anti-phospho ERK1/2, anti-ERK1/2, anti-Histone H3 (Biolegend) and anti-α-Tubulin (B-5-1-2, Sigma) antibodies.

Cells were suspended in ice-cold Lysis buffer (50 mM Tris-HCl, pH 7.5 with 150 mM NaCl, 5 mM EDTA, 1% NP-40, and 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (Nacalai Tesque). Cell lysates were resolved by SDS-PAGE and analyzed by Western blotting as described previously [6] (link). The antibodies used were an anti-FLASH monoclonal antibody (1∶1000; generated in our laboratory [6] (link)), anti-FLASH polyclonal antibody (1∶1000; M-300; Santa Cruz), anti-actin (1∶5000; C4; Chemicon), anti-histone H3 (1∶1000; BioLegend), and anti-estrogen receptor (1∶100; HC-20; Santa Cruz). The secondary antibodies used were Alexa Fluor 488-conjugated anti-mouse or rabbit IgG (1∶5000; Invitrogen).

One day after HCC827 cell seeding at 2.5 × 10⁵ cells/well on a 6-well culture plate, the medium was replaced with medium containing 0–1000 nM Ent. After 4 days, the cells were washed twice with ice-cold phosphate-buffered saline (PBS), collected by trypsinization, washed twice with ice-cold PBS, and lysed with RIPA Buffer (Wako, Osaka, Japan) containing cOmplete Mini, EDTA-free (Roche, Basel, Switzerland) for 30 min on ice. The lysate was centrifuged at 15,000g for 30 min at 4°C and the supernatant was collected. Subsequent processing was carried out as described elsewhere [12 (link)]. Briefly, a 30 μg aliquot of protein was separated on 4–20% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and blotted with primary antibodies [anti-histone H3 (BioLegend, San Diego, CA) or anti-acetyl-histone H3 (Lys9) (Cell Signaling Technology, Danvers, MA)] overnight at 4°C. The next day, the membrane was washed and reacted with goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature. The band intensity was measured using ECL substrate (GE Healthcare, Little Chalfont, Buckinghamshire).