E cad

The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cad, FoxM1, HELLS, Cyclin B1, Cyclin D1, Cyclin A, K14, β-catenin, ZEB1, BrdU, and p63 from Santa Cruz Biotech; GRHL2 (Abnova, Taipei City, Taiwan; H00079977-A01); N-Cad from BD Biosciences (San Jose, CA); FN and Snail from Sigma-Aldrich; p-Smad3 (ser423/425), p-Smad2 (ser465/467), Smad4, p-p38 (Thr180/Tyr182), p-Erk1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p-p65 (Ser536), and Oct-4 from Cell Signaling Technology Inc. (Danvers, MA); hTERT and Sox2 from Abcam (Cambridge, MA); TGF-β from Novus (Littleton, CO); and PCNA from Calbiochem (San Diego, CA). Secondary peroxidase-conjugated anti-rabbit or anti-mouse antibodies were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Tmx and 4-NQO were purchased from Sigma-Aldrich, while TGF-β1 was from PeproTech Inc. (Rocky Hill, NJ). JNK selective inhibitor SP600125 was purchased from Sigma-Aldrich and MEk1/2 inhibitors U0126 and PD98059 were from Cell Signaling Technology Inc.

The following Abs were used: Ecad (sc-7870, Santa Cruz, Heidelberg, Germany), glutathione S-transferase (GST) (MA4-004; Thermo Fisher Scientific, Darmstadt, Germany), α-catenin (GTX22981, GeneTex, Irvine, CA, USA), β-catenin (sc-7199; Santa Cruz; 610153; BD Transduction Laboratories, Heidelberg, Germany), p120-catenin (sc-13957, Santa Cruz), α-tubulin (T9026, Sigma-Aldrich, Munich, Germany), and ZO-1 (61-7300, Thermo Fisher). CD97 was detected using Abs directed to its NTF (H00000976-B01P, Abnova, Taipeh, Taiwan; AF3734, R&D Systems GmbH, Wiesbaden, Germany; 21270971, ImmunoTools, Friesoythe, Germany; HPA013707, Sigma) or CTF (24 (link)).

To investigate the rTsTryp degrading or down-regulating expression of gut epithelium TJs proteins and related pathways, soluble proteins from Caco-2 monolayers pretreated with 20 μg/ml rTsTryp at 37°C for 2 h were analyzed by western blotting as described before [14 (link),37 (link)]. Briefly, Caco-2 cell monolayers were incubated with rTsTryp at 37°C for 2 h, cell proteins were collected, and the cell lysates were separated by 10% SDS-PAGE and transferred subsequently onto PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk in TBST for 1 h at 37°C, and cut into strips. Subsequently, the strips were probed overnight at 4°C with antibodies against ZO-1 (1:1 000, Servicebio, Wuhan, China), E-cad (1:200, Santa Cruz, USA), occludin (2 μg/ml, Santa Cruz), claudin-1 (2 μg/ml, Santa Cruz), PAR2 (1:1 000, Abcam, UK), p-ERK1/2 (1:1 000, Abmart, Shanghai), ERK1/2 (1:1 000, Abmart, Shanghai), GAPDH (1:1 000), and β-actin (1:2 000) (Servicebio). After washing with TBST, the strips were incubated with HRP -conjugated anti-mouse IgG or anti-rabbit IgG as secondary antibodies (1:10 000). Finally, the color of the strips was developed using an enhanced chemiluminescence kit (CWBIO, Beijing, China). The results for the above proteins were normalized using GAPDH as a loading control [4 (link),18 (link)].

Collected A549 and A549/GR cells were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet p-40, 50 mM Tris, pH 8.0, and a protease inhibitor cocktail). Thirty micrograms of each sample was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, USA). Western blotting was performed with rabbit or mouse antibodies against Prx II: LF-PA0091 (AbFrontier, Seoul, South Korea), CD133: PA2049 (Boster Bio, CA, USA), Nanog: sc-293121, OCT3/4: sc-9081, Sox2: sc-365823 (Santa Cruz Biotechnology), VEGFR2: sc-6251, E-cad: sc-7870, Vimentin: sc-6260 (Santa Cruz Biotechnology), Shh: 2207 s, Gli-1: 3538 s, Notch 1: 3268 s (Cell Signaling Technology), CXCR4: YF-MA16239, STAT3: LF-MA30485,pSTAT3 (Tyr 705): LF-PA20474, pSTAT3 (Tyr-727): LF-PA20473 Hes-1: YF-MA11051, and β-Catenin: YF-MA10213 (AbFrontier). Protein expression levels were detected using Super signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, #34577).

After the cell sorting, LCSCs (EpCAM⁺/CD133⁺) and non-stem cells (EpCAM⁻/CD133⁻) were seeded in 24-well plates as 35 × 10³ cells/well. The next day, the cells were fixed with 4% PFA, rinsed with 1X PBS and then permeabilized using a 0.5% TritonX (#28313, Thermo Fisher Scientific). After the cells were incubated with a blocking buffer for 2 h at room temperature, staining was carried out using the following primary antibodies: EpCAM (VU1D9)-Alexa Fluor488 Conjugate (#cs5198, Cell signaling), E-CAD (#sc8426, Santa Cruz) and β-CAT (D10A8) (#cs8480, Cell Signaling). For the F-actin staining, the Phalloidin-iFlour 555 reagent (#ab176756, Abcam) was used. Cells were visualized using a confocal microscope (# LSM880, Zeiss).