β actin

All reagents and solvents should meet the standards of analytical reagent before use, and the melting points of all the synthesized compounds were determined in open capillaries using Shengyan electrothermal PIF YRT-3 apparatus without correction. Bruker AM-400 was applied to record ¹H NMR and ¹³C NMR spectra, and LCQ Deca XP plus was used to determine the ESI/MS spectra. In this study, human cancer cell lines A549, MCF-7, HepG2, Hela and the normal cell WI-38 were purchased from Cell Resources Center of Shanghai Institutes for Biological Science (Chinese Academy of Sciences), which were cultured on the basis of supplier's instructions. DMEM (Dulbecco's modified Eagle medium), FBS (fetal bovine serum) were obtained from Hyclone (Shanghai, China), and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] were provided by Sigma (Beijing, China).
All reagents and solvents are reagent level or are purified by standard methods prior to use. HDAC Inhibitor Drug Screening Kit was purchased from BioVision, and acetyl-histone H3, acetyl-α-tubulin and β-actin were obtained from AFFINITY BIOSCIENCE. Fluor de Lys^® HDAC1 Assay kit (BML-AK511, Enzo^® Life Sciences) and Fluor de Lys^® HDAC6 Assay kit (BML-AK516, Enzo^® Life Sciences) were applied to determine the inhibitory activities of the compounds against HDAC1 and HDAC6 subtypes.

The cells and tissue specimens were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethanesulfonyl fluoride (Beyotime) and a protease inhibitor (CoWin Biosciences). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science and Technology Co., Ltd. Beijing, China). Each sample with equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After running 1–2 h under 120V, the protein was transferred onto a polyvinylidene difluoride membrane. Next, the membranes were blocked with 5% not-fat milk for 1 h at room temperature and incubated with primary antibodies c-Myc, HIF-1α, CXCR4, SDF-1, vimentin, E-cadherin, and β-actin overnight at 4°C (Affinity Biosciences). Subsequently, the membranes were washed 3 times with tris-buffered saline and Tween-20 and incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Beverly, MA, USA). Finally, the membranes were treated with a chemiluminescence reagent and exposed to X-ray. The band intensities were quantified using image J software (National Institutes of Health, Bethesda, MD, USA). Relative protein expression was quantified using control protein β-actin.

20 mg colon tissue was lysed with 200 μL radioimmunoprecipitation (RIPA) lysis solution. The subsequent protocols were performed as previously described [30 (link)]. Briefly, total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA). Membranes were blocked with 5% skim milk and incubated with primary antibodies against zonula occludens-1 (ZO-1, Abcam, USA), occludin (Abcam, USA), and β-actin (Affinity, USA) overnight. Then, horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster, China) were used to incubate the membranes for 2 h. After that, the membranes were visualized with an ECL kit (Boster, China).

For faster and more accurate detection of targeted proteins, we employed the Simple Western™ System (ProteinSimple, USA) for Western blotting. The cell lysis, protein extraction, and quantification methods utilized for each group after transfection were in line with those used in our former research [27 (link)]. Then, boiled protein samples, primary antibodies of MAVS (1:1000, Abcam), β-actin (1:5000, Affinity), Akt (1:1000, Abcam), and Capase-3 (1:5000, Abcam), and the Wes anti-rabbit detection module based on a published manuscript [28 (link)], were added to each well of the Wes Separation 12-230 kDa Capillary Cartridges. All Wes reagents were purchased from ProteinSimple, and the experiment was implemented strictly in compliance with instructions. Eventually, Image J software (version 2.9.0) was adopted for calculating the grey values of the images.

Automated immunoblot was carried out with Simple Wes (Protein Simple, USA) as described by the manufacturer. In brief, 1.5 μg total protein (cell lysate or exosome specimens) was loaded into a Wes assay plate, alongside antibody diluent (Protein Simple); primary antibodies targeting DSP (Santa Cruz, USA), DMP1, CD9, CD63, p-NFκΒ, IκB-a, Myd88, TLR4, IKBKB, BMP2, BMPR1, TGFβ1, TGFβ3, TGFR1, p-Smad2/3, p-Smad1/5/9, Smad4, β-actin, and β-Tublin (Affinity, USA); and anti-rabbit/mouse secondary antibodies (Protein Simple) and streptavidin-HRP. The compass software (Protein Simple) was employed for data analysis.

Cells were washed twice with ice-cold PBS, then lysed in SDS lysis buffer containing 1× protease inhibitor cocktail (Roche Applied Science, Germany). The total protein concentration in the cell lysate solution was then determined via the BCA protein assay (Thermo Fisher Scientific, USA). Protein samples (40 μg) were separated by electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). After being blocked with 5% skim milk (BD Biosciences, USA) for 1 h, the membranes were incubated with specific primary antibodies; β-actin (Affinity Biosciences, USA, T0022; 1:4000), GAPDH (Immunoway Biotechnology, USA, YM3040; 1:4000), YBX1 (Santa Cruz Biotechnology, USA, sc-101,198; 1:1000), G3BP1 (Santa Cruz, sc-98,561; 1:1000), SPP1 (Abcam, USA, ab-69,498; 1:2000), p-p65 (Cell Signaling Technology, USA, #3033; 1:1000), and p65 (Cell Signaling Technology, USA, #8242; 1:1000), overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP-conjugated anti-mouse (Affinity, S0002) or anti-rabbit (Affinity, S0001) secondary antibodies for 1 h and visualized with ECL system (Millipore, USA).

The prostate protein levels were analyzed by Western blotting. In brief, total protein was extracted by RIPA Lysis Buffer and separated by polyacrylamide gels, and then, was transferred onto PVDF membranes. After blocked with 5% BSA solution, the membranes were incubated with primary antibodies, including GAPDH (T0004, dilution 1 : 5000; Affinity), β-actin (AF7018; dilution 1 : 5000; Affinity), IL-1β (AF7018; dilution 1 : 5000; Affinity), and TNF-α (ab6671; dilution 1 : 1000; Abcam). Then, the membranes were incubated with secondary antibodies for 1 hour at room temperature. Finally, immunodetection was performed using Chemi-Doc Imaging System (Bio-Rad, Hercules, CA, USA).