P histone h3

The 786-O and SN12-PM6 cells were each cultured in three wells of a 24-well plate with coverslips. Following transfection with lentivirus for 24 h and screening with puromycin for a further 48 h, the cells were fixed and then blocked in 3% goat serum albumin-PBS. Primary antibody against γ-H2AX (1:100, 9718s, Cell Signaling Technology, Inc.) and p-histone H3 (Ser10, 1:800, cat. no. 3377, Cell Signaling Technology, Inc.) was incubated with the cells overnight at 4°C. The cells were then washed and incubated with Alex Fluor 594 goat anti-rabbit IgG (1:500, A32740, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The coverslips were then placed on fluorescent mounting medium with DAPI (ZLI-9557, OriGene Technologies, Inc.) and incubated at room temperature. The slides were imaged using a Leica SP5 confocal microscope (Leica Microsystems GmbH) at ×100 magnification and analyzed using software (Leica LAS AF Lite 2.6; Leica Microsystems CMS GmbH).

The spleens, lungs, and livers were obtained from Hmox^−/−and CreLyz:Hmox1^flfl mice and either snap frozen in freezing medium followed by cutting into 6 μM sections or fixed in formalin and processed for paraffin embedding and antigen retrial using citrate buffer as previously described.^{33 (link)} Bones were fixed, decalcified, and paraffin embedded. After fixation with 2% PFA, sections were permeabilized with 0.5% Triton X-100, blocked with horse serum, and respective primary antibodies were applied over night. The following day, secondary antibodies conjugated with Alexa-488 were applied for 1 h at room temperature. Nuclear staining was done with Hoechst, and slides mounted on gelvatol. Pictures were taken at 20 or × 40 magnification under confocal microscopy. The following primary antibodies were used: CD14 (BD Biosciences, San Jose, CA, USA), CD133 (Sigma-Aldrich), HO-1 (Epitomics), P-Histone-H3 (Cell Signaling), F4.80 (Serotec).

Cells were lysed on ice in RIPA buffer (Thermo Scientific, J62524) containing protease inhibitor (Thermo Scientific, Pierce A32965) and phosphatase inhibitor (Thermo Scientific, Pierce A32957). Lysed cells were spun down and supernatant was incubated with NuPAGE^™ LDS sample buffer (Invitrogen, NP0007) for 5 minutes at 95 °C. Proteins were separated using NuPAGE^™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels (Thermo Fisher Scientific, NP0321BOX) in NuPAGE^™ MES SDS Running Buffer (Thermo Fisher Scientific NP0002), followed by transfer onto nitrocellulose membranes (BIO RAD, 1620115) using Towbin Buffer (2.5 mM Tris, 19.2 mM glycine, pH 8.3) containing 20% methanol. Membranes were blocked with blocking buffer (3% milk, TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5)) for 30 minutes at room temperature, followed by incubation with primary antibody in 1% milk and in TBST buffer overnight (16 hour) at 4 °C. Membrane was washed 3 times in TBST buffer for 10 minutes and incubated with secondary IRDye antibodies (1:5000 LI-COR) in blocking buffer (1% milk, TBST, 0.001% SDS) for 30 minutes at room temperature. Membranes were washed 3 times in TBST buffer and scanned on an Odyssey CLx Imaging System (LI-COR). The following antibodies were used: ZAG (Santa Cruz, sc-13585 1:200), P-Histone H3 (Cell signaling, 34655 1:1000) IRDye 680 RD (LiCor, 926-68072 1:2000).