H4k16ac

To obtain nuclear protein extracts from MSCs, 24 h after culture on substrates with distinct stiffness, cells were washed with PBS and then scraped in Laemmli buffer. The subcellular fractionation was done following the protocol described in^{75 (link)}, and nuclear fractions were collected into microtubes and then heated at 95 °C for 5 min. Next, the samples were spun down and passed ten times through a 25 G needle and further analyzed by Western blot. Proteins were separated by SDS-PAGE [12.5% (w/v) acrylamide–bisacrylamide (Bio-Rad) gels] and transferred onto PVDF membranes that were subsequently probed with specific antibodies against H4K16ac (Abcam) and H4 (Cell Signaling) followed by the incubation with the respective alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch). The membranes were incubated for 5 minutes with enhanced chemifluorescence substrates (ECF – GE Healthcare) and imaged in a Molecular Imager FX Pro Plus system (BioRad) using the Quantity One software (BioRad). The acquired images were analyzed with Image Lab software, version 5.1 (BioRad).

Different cells were harvested after treatment and proteins were extracted to detect the expression by Western blotting as previously described with minor modifications [46 (link)]. Equal amounts of proteins were size fractionated by 9 to 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Anti-SIRT6 (2590 S, Cell Signaling Techology, Danvers, MA, USA), anti-KAT8 (ab200660, abcam, Cambridge, MA, USA), anti-Acetyl-lysine (PTM-105RM, PTMBIO, China), anti-Flag (F1804, Sigma Aldrich), anti-SIRT1 (8469 S, Cell Signaling Techology, Danvers, MA, USA), anti-SIRT7 (5360 S, Cell Signaling Techology, Danvers, MA, USA), H4K16ac (ab109463, Abcam Cambridge, MA, USA), anti-RNA Pol II (sc-47701, Santa Cruz, CA, USA), anti-E-cadherin (14472 S, Cell Signaling Techology Danvers, MA, USA), anti-N-cadherin (13116 S, Cell Signaling Techology Danvers, MA, USA), anti-Histone H4 (ab177840, Abcam, Danvers, MA, USA), anti-His (PM032, MBL), anti-GST (sc-138, Santa Cruz, CA, USA), anti-GCN5 (sc-365321, Santa Cruz, CA, USA), anti-myc (M047-3, MBL), anti-α-tubulin (BE0031, EASYBIO, Beijing, China) and anti-β-actin (4967, Cell Signaling, Danvers, MA, USA) were used and the blots were developed using an enhanced chemiluminescence kit (Amersham Corp.).

To carry out Western blotting experiments, different concentrations (0.5, 1.0, and 2.0 mg L^-1) of AgNPs were used. DI water-suspended yeast cells with different doses of as-prepared AgNPs were incubated for 3 h (200 rpm, 30°C). Each experiment was performed in triplicate under similar culture/treatment conditions. Protein cell extraction from 20% trichloroacetic acid precipitation and further immunoblotting analysis were carried out in accordance to a procedure as described in our previous article (Babele et al., 2018 (link)). Primary antibodies general H3 (Sigma, H0164), H4 (Sigma, SAB4500312) H3K4me2 (Abcam, 8895), H3K4me3 (Abcam, 32356), H3K56Ac (Sigma, SAB4200328), H4K8Ac (Abcam, 46982), and H4K16Ac (Abcam, 61240) were used to investigate global histone modifications. The same blots were re-probed with anti-Tbp, -H3, and -H4 protein and used as loading control; purified H3 and H4 proteins were used as marker to represent the correct bands (Figure S4). The detection of proteins was carried out using a relevant secondary antibody, IRDye^® 800CW goat anti-rabbit IgG or anti-mouse IgG (1:15,000; LI-COR Biosciences). An Odyssey infrared imager (LI-COR Biosciences) was used to scan the blots, and as-received images from three independent performances for each condition are shown.

Total proteins were harvested from BMMSCs, bone marrow, and other organs with RIPA lysis buffer (Beyotime, China) and quantified by BCA assay. Next, the proteins were separated on sodium dodecyl sulfonate-polyacrylamide gels (SDS-PAGE), transferred to PVDF membranes (Millipore, Billerica, MA, USA), blocked in TBST containing 5% BSA, and incubated in first antibodies with Beclin1 (Cell Signaling, 3738, 1:1000), ATG7 (Cell Signaling Technology, 8558, 1:1000), LC3 (Cell Signaling Technology, 1274, 1:1000), p62 (Proteintech, 18420-1-AP, 1:1000), HDAC9 (Abcam, ab59718, 1:1000), H3K9ac (Abcam, ab10812, 1:1000), H3K18ac (Cell Signaling Technology, 9675, 1:1000), H4K16ac (Abcam, 13534, 1:1000), H3 (Cell Signaling Technology, 9715, 1:1000), p53 (Cell Signaling Technology, 2524, 1:1000), phospho-p53 Antibody (R&D systems, AF1043, 1:2500), Runx2 (Cell Signaling Technology, 2435, 1:1000), ALP (R&D systems, AF2910, 1:500), PPAR-γ (Abcam, 2435, 1:300), and GAPDH (Cwbiotech, CW0100, 1:4000), respectively. Then, the membranes incubated in secondary antibodies which coupled to peroxidase (Cwbiotech, China). Finally, the signals were detected by an enhanced chemiluminescence kit (7seapharmtech, China).

Cells were fixed (paraformaldehyde), permeabilized (Triton X-100), blocked (BSA), and stained with antibodies against H4K16ac (Abcam), 53BP1 (Novus Biologicals), and the Xpress-tag encoded by His-TPX2 as per standard laboratory procedures. Nuclei were counterstained with DAPI. Images were acquired with a Nikon Eclipse TE2000-E confocal microscope.