Ab182651

The tumor sections of liver cancer xenografts from nude mice (5 μm thick) were stained with anti-PI3K antibody (1:1000; ab182651; Abcam), anti-p-AKT (1:500; ab8933; Abcam), Cleaved Cas-3 (1:20; ab2302, Abcam) and anti-Ki67 antibody (1:500; ab15580; Abcam) at 4°C overnight, and then reacted with anti-IgG secondary antibody (1:1000; ab6721; Abcam) for 30 min. 3,3-diaminodiphenylamine (DAB, DA1010, Solarbio, Beijing, China) was used for visualization. Five fields of 200× magnification were randomly captured at each repeat using an inverted microscope (Nikon, Tokyo, Japan).

Following treatment of U87 cells with Lid-FA-Lip (1 mM lidocaine, 37˚C for 24 h), total protein was extracted with RIPA buffer (Beyotime Institute of Biotechnology) and protein determination was performed using the BCA method. Protein samples (10 µg loaded per lane) were separated on 10% SDS-PAGE and transferred onto PVDF membranes (250 mA; 2 h), which were then blocked with 5% milk in TBS with 0.05% Tween-20 at 25˚C for 2 h. The PVDF membranes were subsequently treated with primary antibodies targeting the following proteins (all from Abcam): Bcl-2 (ab32124; dilution, 1:500), matrix metalloproteinase 2 (MMP2; ab92536; dilution, 1:500), Ki67 (ab92742; dilution, 1:1,000), phosphorylated (p-)PI3Kp85 (phospho Y607; ab182651; dilution, 1:1,000), PI3Kp85 (ab135253; dilution, 1:500), p-AKT (phospho T308; ab38449; dilution, 1:1,000), AKT (ab18785; dilution, 1:1,000) and GAPDH (ab8245; dilution, 1:3,000) at room temperature for 2 h. The membranes were then incubated with anti-rabbit (cat. no. ab6271; Abcam) or anti-mouse (cat. no. ab6728; Abcam) HRP-conjugated secondary antibodies (1:5,000 dilution) at room temperature for 1 h. Signals were then visualized using an ECL kit (Novex™ ECL Chemiluminescent Substrate Reagent kit; Thermo Fisher Scientific, Inc.) and visualized by ImageJ 8.0 software (National Institutes of Health).

Cells were lysed in RIPA Lysis Buffer and a BCA Protein Assay kit (Thermo Fisher Scientific) was used to determine the protein concentration. Proteins of each sample were separated by SDS-PAGE gel. Then, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% non-fat milk, followed by the incubation with primary antibodies at 4°C overnight: anti-Bax (Abcam; ab32503) (1:1000), anti-active caspase 3 (Abcam; ab2302) (1:1000), anti-Bcl-2 (Abcam; ab32124) (1:1000), anti-β-actin (Abcam; ab8227) (1:1000), anti-PTEN (Abcam; ab32199) (1:1000), anti-p-PI3K p85 (p-p85, Abcam; ab182651) (1:1000), anti-p-Akt (Abcam; ab38449) (1:1000). The membranes were washed in TBST three times then incubated with secondary antibodies for 1 hr at room temperature. Chemiluminescence (Millipore Corporation, Billerica, MA, USA) were applied to measure protein expression using densitometry analysis (ImageJ software).

AS-IV (HPLC ≥ 98.81%) and fine particulate matter standards were respectively purchased from Chengdu Munster Company (China) and National Institute of Standards and Technology (USA). 3-Methyladenine (3-MA) was purchased from Selleck, and polyvinylidene fluoride membranes were purchased from Bio-Rad (USA). Antibodies against the following targets were obtained from Abcam (Cambridge, UK): LC3B (ab48394), p62 (ab56416), PI3K (ab182651), Akt (ab185633), p-Akt (ab38449), mTOR (ab2732), p-mTOR (ab137133), Lamin B (ab16048), and GAPDH (ab181602). An antibody against p-PI3K (AF3242) was obtained from Affinity Biosciences (USA). An antibody against p65 (8242S) was obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (GB23303) and 4°C tissue radioimmunoprecipitation assay lysates were obtained from Servicebio (Wuhan, China).