Goat serum

IR-induced 53BP1 kinetics were determined as previously outlined with modifications (19 (link)). Cells were grown on coverslips one day before the experiment and on the day of the experiment, cells were exposed to 1Gy of γ-rays. At different time points after IR (see figure), cells were washed twice with ice cold 1× PBS and fixed with 4% paraformaldehyde (in 1× PBS) for 20 min at RT, washed 5 times with 1× PBS, and incubated in 0.5% Triton X-100 on ice for 10 min. Cells were washed 5 times with 1× PBS and incubated in blocking solution (5% goat serum (Jackson Immuno Research) in 1× PBS) overnight. The blocking solution was then replaced with the 53BP1 (SC-22760, Santa Cruz) primary antibody diluted in 5% goat serum in 1x PBS and the cells were incubated for 2 h. Cells were then washed 5 times with wash buffer (1% BSA in 1× PBS). Cells were incubated with the Alexa Fluor 488 (1:1000) (Molecular Probes) secondary antibody in 1% BSA, 2.5% goat serum in 1× PBS for 1 h in the dark, followed by five washes. After the last wash, cells were mounted in VectaShield mounting medium containing DAPI. The images were acquired using a Zeiss Axio Imager fluorescence microscope utilizing a 63× oil objective. ≥100 cells were analyzed for each time point.

For immunohistochemical staining, 4-μm-thick sections received antigen retrieval and endogenous peroxidase activity blocking. Then, the samples were incubated in goat serum for 30 min at room temperature to block the nonspecific antigen, followed by incubating in primary antibodies against K10 (1:200; Santa Cruz), K14 (1:200; Santa Cruz), and p-AKT (1:250; Affinity Biosciences) overnight at 4 ℃. Finally, the immune reactivities of the sections were determined using the HRP-streptavidin detection system (ZSGB-bio, Beijing, China). Images from above-mentioned sections were acquired with a BX63 fluorescence microscope (Olympus).
For immunofluorescence staining, 4-μm-thick sections received antigen retrieval and permeabilizing with 0.5% Triton X-100. After blocking with goat serum (Solarbio), primary antibodies against Ki67 (1:250; Affinity Biosciences), Cytokeratin (Pan) (1:200; Huabio, Hangzhou, China), E-cad (1:200; Affinity Biosciences), Vim (1:500; Abcam), and N-cad (1:200; Santa Cruz) were loaded and kept overnight at 4 ℃. Then, Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Abcam) were loaded at room temperature for 2 h. The nuclei were stained with DAPI (Beyotime), with the images visualized via the LSM-980 confocal microscope (Carl Zeiss).

The rabbit anti-CD146 polyclonal antibody and mouse anti-CD146 monoclonal antibody AA1 and AA4 were generated in our laboratory (Zhang et al., 2008 (link)). Rat anti-mouse CD146 (clone ME-9F1) was purchased from BD Biosciences. Anti-mouse Tek-PE and anti-mouse CD31-APC antibodies were purchased from Tianjin Sungene Biotech Co., Ltd. Antibodies specific for phospho-p38 MAPK, p38 MAPK, phospho-NF-κB p65 (Ser536), phospho-ERK, ERK, AKT, were purchased from Cell Signaling Technology. Antibodies specific for I-κB were from Beijing Zhong Shan-Golden Bridge Biological Technology CO., LTD. Antibodies against phosphor-VEGFR-2 (Tyr 1214) and phospho-AKT (Ser 473) were purchased from Signalway Antibody. Antibodies specific for CD31 and GAPDH were purchased from Abcam. HRP-conjugated goat anti-mouse or rabbit IgG were purchased from GE Healthcare. Enhanced chemiluminescence assay kits were purchased from Pierce. Biotin-conjugated secondary antibodies and HRP-conjugated streptavidin were purchased from Dianova. Goat serum and the DAB substrate system were purchased from Santa Cruz Biotechnology. DAPI was purchased from Roche. Fluorescein isothiocyanate-dextran was purchased from Sigma. Growth factor-reduced Matrigel and collagenase were purchased from BD Biosciences. VEGF-A was purchased from Upstate Biotechnology.

Graphene oxide nano-colloids (GON) dispersed in H₂O (2 mg/mL), paraformaldehyde (PFA), methanol and all the reagents used for solutions were purchased from Sigma Aldrich. Bovine Serum Albumin (BSA) and goat serum were purchased from Santa Cruz Biotechnology. The signaling pathway inhibitors Dabrafenib/GSK2118436 (DAB) and Trichostatin A (TSA) were purchased from Selleckchem (www.Selleckchem.com).