Ab1667

The colon sections were deparaffinized in xylene and rehydrated into water through graded alcohol. For immunohistochemistry, the sections were blocked with 5% BSA and incubated with the anti-Ki67 antibody (1:100, ab1667, Abcam, Cambridge, United Kingdom) and anti-E-cadherin (1:100, 3195S, Cell Signaling Technology, Danvers, MA, United States) as the primary antibodies to detect the antigen overnight at 4 C. Then, the primary antibody was combined with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and the signals were detected using diaminobenzidine. Images were acquired using NanoZoomer 2.0-HT microdissection systems (Bensons, Japan) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, United States). For immunofluorescence, the colon tissue sections were incubated with primary antibodies anti-cleaved caspase-3 (1:100, 9661S, CST, Danvers, MA, United States) and anti-caspase-1 (1:100, 8932S, Abclonal, Wuhan, China) overnight at 4°C. The sections were incubated with the Alexa Fluor^® 488-conjugated anti-rabbit secondary antibodies (1:400, GB35303, Servicebio, Wuhan, China) at room temperature and then counterstained with DAPI (ab104139, Abcam, Cambridge, United Kingdom) to stain the nuclei. Images were collected on a microscope imaging system (Nikon DS-U3, Tokyo, Japan).

IHC was conducted using protocols described previously [19 (link)]. Briefly, sections were incubated with primary antibodies against 5mC (ab10805, Abcam, Cambridge, UK), p-mTOR (ab109268, Abcam), DNMT1(5032S, CST, Danvers) and Ki67 (ab1667, Abcam). The immunostaining intensity was given a score of 1–3 (1, negative or weak; 2, moderate; 3, strong), and the percentage of positive immunostaining was scored as 0–100%. The percentage and intensity score were multiplied to obtain the total immunohistochemistry score (range of 0–300) of 5mC and p-mTOR. The IHC score of DNMT1 and Ki67 were obtained by multiplying the percentage of positive cells and 100 (range of 0–100).

We lysed the cells to obtain the total protein using RIPA lysis buffer (R0013D, Beyotime), and then used a BCA kit to detect the total protein concentration. 50 μg total protein was analyzed by 10% SDS-PAGE. After transferring to the PVDF membrane and sealing with 5% skimmed milk, the primary antibodies against Bax (ab32503, Abcam), Bcl2 (ab182858, Abcam), or PCNA (A12417, Abclone), or Ki-67 (ab1667, Abcam), or p-PI3K (ab278545, Abcam), or PI3K (ab140307, Abcam), or p-AKT (4060, Cell Signaling Technology), or AKT (4685, Cell Signaling Technology) were incubated overnight at 4°C. After being incubated with a secondary antibody at room temperature for 1 hour, the proteins were visualized with ECL solution (WBKLS0100, Beijing Xinjingke Biotechnologies Co., Ltd., China), followed by densitometry analysis using Image J 3.0 (IBM, USA). β-Actin was loaded as control.

Ki67 and Vimentin protein expression in tissue samples were determined by IHC as described in the previous method [29 (link)]. The carcinoma and non-cancerous tissue sections were randomly selected and fixed in acetone for 10 min at − 20 °C, permeabilized with 0.2% Triton (Sigma) for 10 min at room temperature, incubated with a blocking solution (3.75% BSA/5% goat serum, Zymed, Carlsbad, CA, USA) for 30 min, and incubated for 2 h with anti-Vimentin (1:200, ab92547, Abcam), anti-Ki67 (1:150, ab1667, Abcam) and anti-ZEB1 (1:100, ab203829, Abcam). All sections were incubated with goat-anti-rabbit HRP-labeled secondary antibodies, then incubated in DAB reagent (Beyotime, China), and subsequently stained with hematoxylin (Beyotime, China). The sections were observed under microscopy (Olympus, Tokyo, Japan).

Recruited Ly6C^high EdU positive cells in the aortic root plaques were detected using Click-iT EDU Imaging Kit (MP 10338, Invitrogen). Recruitment of Ly6C^low beads were counted in the plaques using a fluorescent microscope, EVOS FL COLOR (Thermo Fisher Scientific). Apoptosis in the plaque was analyzed by staining the plaques with anti-cleaved caspase 3 (1:100, 9664, Cell signaling). Cell proliferation was analyzed by staining the plaques for Ki67 (1:100, ab1667, Abcam). Alpha smooth muscle actin (smooth muscle cells marker) was measured with SMaA-AF488 (1:100, 53-9760-82, Invitrogen). Aortic root plaques were also stained for DAPI (ProLong™ Gold Antifade Mountant with DAPI, P36931, Thermo Fisher Scientific). Number of positive cells was measured for each mouse.