As011

The tumor tissues from each mice group were broken into pieces of 200–250 mm³ using the homogenizer. Then, RIPA buffer was added to destroy the cells and incubated for 4 h. Supernatants were harvested and clarified by centrifugation at 12,000 × g for 3 min. The loading buffer diluent was added to make the total protein content consistent in the three groups. The samples were boiled in the water bath for 10 min and stored at −80 °C. The proteins were dissociated by 15% SDS-PAGE. The gel was transferred onto PVDF membranes and blocked by 5% BSA for 1 h. Subsequently, LC3B protein rabbit pAb (1:500 A5601, ABclonal) was employed at 4 °C overnight. The following day, membranes were incubated with appropriate secondary antibodies (1:500; AS011, ABclonal) for another 1 h. All the membranes were rinsed using TBST for 15 min three times. Finally, the target bands were exposed and visualized using the ECL detection system (Bio-Rad, USA). Besides, the internal reference was set by GAPDH.

The rat hippocampus section was pre-incubated with 0.3% Triton X-100 in PBS for 10 min and blocked with 0.1%Triton X-100 for 1 h. Primary rabbit anti-NeuN (1:500, ab177487, Abcam) and secondary goat anti-rabbit immunoglobulin G (IgG; 1:100, AS011, ABclonal, USA) were employed for immunofluorescence. The samples were placed in a DAPI matrix for nuclear staining and examined by inverted microscopy (Olympus IX71, Tokyo, China) in five randomly selected fields of the hippocampus CA1 region. Positive NeuN cells were counted by ImageJ software.

The western blotting assay was performed to detect the expression of the Khib proteins in PC tissues and cell lines. Briefly, cells or tissues were lysed by pre-formulated lysis buffer (RIPA, 1% Protease Inhibitor Cocktail, 3 μM TSA, and 50 mM NAM). The protein concentrations were measured by BCA kit (P0012S; Beyotime Biotechnology, China), and then we analyzed 50μg protein of every sample on 10% SDS-PAGE gels. All blots were visualized using ECL Kit (RM00020P; ABclonal, Wuhan, China) and the intensity of the bands was assessed by Image Lab (Bio-Rad, California, USA). The antibodies used in this study were: pan-lysine 2-hydroxyisobutyrylation antibody (1:1000; PTM-802; PTM Bio, Hangzhou, China), FITC antibody (1:50; AS011; ABclonal, Wuhan, China), β-actin antibody (1:1000; AC026; ABclonal, Wuhan, China). β-actin was used as a loading control and the intensity of the entire bands was assessed with ImageJ2 (National Institute of Mental Health, Bethesda, MD, USA). All experiments were performed three times following the same procedure.

Briefly, frozen artery tissues were sliced into 4–6 μm sections. The slides were then blocked with 5% BSA for 1 h. After that, primary antibodies against SRPK1 (1:100 dilution, A12510, ABclonal, China), ZNF281 (1:100 dilution, A12650, ABclonal, China), S100A8 (1:100 dilution, A15315, ABclonal, China), and S100A9 (1:100 dilution, A9842, ABclonal, China) were incubated overnight at 4°C. After washing with TBS, the cryosections were incubated for 1 h with the secondary antibody (goat anti-rabbit antibody, 1:100 dilution, AS011, ABclonal, China). The sections were visualized using a fluorescence microscope. Images were analyzed by Image J software (Image J 1.53, NIH, USA).

The detailed procedures have been described in the previous study.¹³ Briefly, tissues were sliced into 5‐μm‐thick frozen sections. Next, after blocking for 1 hour with 5% bovine serum albumin, primary antibodies, including rabbit anti‐MSR1 (ABclonal, A2401), mouse anti‐IBA1 (Abcam, ab283319), rabbit anti‐CD86 (ABclonal, A19026) were incubated with the samples overnight at 4°C. Then, the primary antibodies were washed three times by TBS. Secondary antibodies, including the goat anti‐rabbit antibody (AS011, ABclonal) and the goat anti‐mouse antibody (AS001, ABclonal) were placed on the slides and incubated for 1 hour at 25°C. The antibody dilution ratio was following the online instructions. The sections were subsequently stained with DAPI for nuclear labelling. Images were analysed by Image J 1.4 software.

Cells were cultured on coverslips and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Cells were permeated by PBS supplemented with 0.2% Triton X-100 for 10 min at room temperature and blocked by PBS supplemented with 5% BSA and 0.2% Triton X-100 for 60 min at room temperature. The following primary antibodies were used: anti-BRD4 (Abcam, ab128874, 1:200 dilution), anti-MED1 (Abcam, ab64965, 1:200 dilution), anti-p300 (Abcam, ab259330, 1:200 dilution), anti-Cyclin T1 (Abcam, ab184703, 1:200 dilution). Antibodies were diluted in a blocking buffer and incubated overnight at 4°C. After washing three times with PBS for 5 min each. Fluorescent secondary antibody (ABclonal, AS011, 1:500 dilution) was incubated by PBS supplemented with 0.2% Triton X-100 for 60 min at room temperature in dark. The cells were washed with PBS three times for 5 min each. 4,6-diamidino-2-phenylindole (DAPI) (Beyotime, C1005) was used to stain nuclei for 5 min at room temperature in the dark. Coverslips were mounted onto microslides (CITOTEST, 1A5101). Coverslips were sealed with transparent nail polish and stored at −20 °C. Images were acquired at Zeiss LSM980 Airyscan2. Images were processed and analyzed using Imaris v9.9.

Tissue microarrays (DC-Pro11018, Xian, China) including 74 prostate cancer tissues and 6 non-cancer prostate tissues, along with associated detailed clinical information, were purchased from Alenabio Biotech (Xian, China). Clinical information for the prostate cancer tissue microarray is shown in Supplementary Table 5. The sections were incubated overnight at 4°C with antibodies against anti-CD163 (rabbit, 1:100, A8383, Elabscience, Wuhan, China) and anti-PLK1 (mouse, 1:50, TA500393S, ORIGENE, Rockville, USA). The sections were then washed three times with cold PBS and stained with Cy3 Goat Anti-Mouse IgG (H+L) (1:100, AS008, ABclonal, Wuhan, China) or FITC Goat Anti-Rabbit IgG (H+L) (1:100, AS011, AS008, ABclonal, Wuhan, China) secondary antibodies. Nuclei were stained with DAPI. Stained tissues were visualized using an Olympus IX73 microscope (Waltham, MA). IOD (integral optical density) was calculated using ImageJ (1.46r, National Institutes of Health, USA).