Snail slug

PLGA, PFOB, tetraethoxysilane (TEOS), and (3-aminopropyl) triethoxysilane (APTES) were bought from Aladdin Company (Shanghai, China). PTX was bought from Macklin Company (Shanghai, China), and ICG was bought from Sigma-Aldrich Company (St. Louis, MO USA). Commercial Gd-DTPA was bought from Shering AG (Berlin, Germany). Targeting ligands uPA and RGD were bought from Sigma-Aldrich Company (St. Louis, MO USA) and GL Biochem Ltd. (Shanghai, China), respectively. Fluorescent probes DHE and DPBF were bought from Sigma-Aldrich Company (St. Louis, MO USA). Antibodies against HIF-1α, Vimentin, Snail-Slug, and E-cadherin were bought from Abcam (Cambridge, UK). The water utilized in this study was distilled.

Total proteins were extracted from cells, and western blotting was performed as previously reported^{33 (link)}. The primary antibodies used were: PCNA (Cell Signaling Technology, Danvers MA, USA; #13110; dilution 1:5000); Hexokinase II (EPR20839; 1:500), HIF1α(1:1000), GLUT1 (1:2000), N cadherin (5D5; 1:1000), and Snail/Slug (1:1000), all procured from Abcam (Cambridge, UK); and β-actin-HRP (Santa Cruz Biotechnology, Dalla TX, USA; C4; dilution 1:1000). Western blotting experiments from biological replicates showed similar expression data, attesting to the reproducibility of the results. For band quantification, images were analyzed using Image Lab software (Bio-Rad, Hercules, California, USA). For band quantification, images were analyzed using IMT i-Solution software Martin Microscope Company, Easley, USA).

DMEM-F12 medium and foetal bovine serum (FBS) were purchased from the Gibco (Invitrogen, USA). Penicillin, streptomycin and BCA protein assay kits were purchased from the Solarbio (Beijing, China). Polystyrene plate for transwell was purchased from the Corning (New York, USA). The primary antibodies were diluted 1:2000 before use, including OLA1 (Cat. #ab229090, Abcam), GAPDH (Cat. #ab9485, Abcam), GAPDH (Cat. #ab8245, Abcam), N-cadherin (Cat. #ab18203, Abcam), E-cadherin (Cat. #ab76055, Abcam), Snail (Cat. #PA5–11923, Invitrogen), Snail + Slug (Cat. # ab180714, Abcam), Zeb-1 (Cat. # ab155249 Abcam) and SMAD2 (Cat. #5339, Cell Signaling Technology), p-SMAD2 (Cat. #18338, Cell Signaling Technology), SMAD4 (Cat. #9515P, Cell Signaling Technology). TGF-beta 1 antibody (Cat. # 21898–1-AP) was purchased from ProteinTech (Wuhan, China). All the chemical compounds were analytically pure reagents.

HCC cells were lysed in ice-cold RIPA lysis buffer with protease inhibitor and phosphatase inhibitor. Protein concentration was determined by using the BCA method. Thirty micrograms of total protein was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked in 0.5% BSA for 1 h at room temperature and incubated overnight with antibodies to PTEN, FOXO3, PI3K, AKT, p-AKT, ERK1/2, p-ERK1/2, C-myc, active caspase3, Bcl2, Bax, E-cadherin, TIMP1, MMP2, snail/slug, twist, and GAPDH (Abcam, USA). Then, the membranes were incubated with the corresponding HRP-labeled secondary antibodies for 1 h at room temperature. Chemiluminescent signals were captured by the Imager (Tiangen, China).

Cells treated with UA or KU for 24 h were washed twice with ice-cold PBS and lysed in lysisbuffer^{26 (link)}. Antibodies against E-cadherin (61018, BD Biosciences, San Diego, CA, USA), N-cadherin (610921, BD Biosciences), Snail/Slug (ab180714, Abcam, Cambridge, MA, USA), Twist (ab49254, Abcam), PARP (9542, Cell Signaling), Caspase-3 (9662, Cell Signaling), α-Tubulin (2125, Cell Signaling), and ZEB2 (HPA003456, Sigma) were detected with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) with the use of Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA) and luminescence imaging (Image Quant LAS 4000 mini). Multi-Gauge 3.0 was used to measure bands, and relative density was calculated based on the density of the α-Tubulin bands in each sample. Expression of values were as arbitrary densitometric units corresponding to signal intensity.

The whole-cell lysates were generated using the Whole Cell Lysis Assay Kit (KeyGEN, China) according to the manufacturer’s protocol. The protein concentration was detected by the BCA method (Thermo Fisher Scientific, USA). β-Actin (1: 1000, Abcam, UK) was used as an internal control. Primary antibodies included ISL1 (0.5 μg/mL, Developmental Studies Hybridoma Bank, USA), GATA3 (1:1000, Invitrogen, USA), E-cadherin (1:10,000; Abcam, UK), N-cadherin (1:5000; Abcam, UK), vimentin (1:1000; Abcam, UK), snail + Slug (1 μg/mL; Abcam, UK), Bcl2 (1:1000; Abcam, UK), Bax (1:1000; Abcam, UK), Activated Caspase3 (1:1000; Abcam, UK), AKT (1:1000, Cell Signaling Technology, USA), p-AKT (1:1000, Cell Signaling Technology, USA), and AURKA (1:1000; Abcam, UK). Secondary antibody included goat anti-rabbit antibody (1:10,000, Jacson ImmunoResearch, USA) or goat anti-mouse antibody (1:10,000, Jacson ImmunoResearch, USA). The ECL chemiluminescence detection system (Bio-Rad, USA) was used for signal detection.

Immunohistochemistry (IHC) was performed the same as standard procedures. Briefly, 5 mm tissue sections were deparaffinized in xylene and rehydrated through a gradual decrease in the concentration of ethanol. The antigen epitopes were then unmasked using sodium citrate buffer (pH 6.0) by a standard microwave heating technique. After hydrogen peroxide blocking and normal serum blocking, Ig Blocking Reagent (Vector Laboratories, Burlingame, CA, USA) was used as a blocking buffer. Subsequently, the sections were incubated overnight at 4 °C with the following primary antibodies: CD44 (abcam, ab24504), GFP (cell signaling, #2956), Ki67 (abcam, ab66155), Snail/Slug (abcam, ab180714), CK19 (abcam, ab15463), E-Cadherin (cell signaling, #3195), Vimentin (abcam, ab45939) and N-cadherin (abcam, ab18203). After primary incubation, sections were incubated with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), followed by incubation with the ABC reagent (Vector Laboratories, Burlingame, CA, USA). Detection was accomplished using DAB (3,3’-diaminobenzidine) substrates (Vector Laboratories, Burlingame, CA, USA). Incubation of sections with phosphate-buffered saline (PBS) served as negative controls. Sections were lightly counter-stained with hematoxylin and mounted. The IHC-stained slides were mounted in Micromount (Leica, Nussloch, Germany).