2.1. Reagents
Chloroquine (CQ), cycloheximide (CHX) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the purest grade available.
2.2. Cell culture
HCT-116 (CCL-247) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Caco-2 cells were obtained from Korean Cell Line Bank (Seoul, South Korea). HCT-116 and Caco-2 cells were cultured in RPMI-1640 and Minimal Essential Medium (MEM), respectively, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
2.3. Plasmid transfection
The colon cancer cells were transiently transfected according to the manufacturer's instructions with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Transfection was performed as previously described [15]. The Rab25 cDNA was subcloned into a pcDNA3 vector, and an empty pcDNA3 vector was used as a negative control. RAS-V12 constructs were kindly provided by Dr. A.R Moon (Duksung University, Seoul). Claudin-7 constructs were purchased from Origene (Rockville, MD, USA). siRNAs against Rab25 (SASI_Hs01_00216284), claudin-7 #1 (SASI_Hs01_00214821), claudin-7 #2 (SASI_Hs01_00214822) were purchased from Sigma-Aldrich (St Louis, MI, USA). Control scrambled siRNA was from Invitrogen (Carlsbad, CA, USA).
2.4. Quantitative RT-PCR (qRT-PCR)
Isolation of total RNA and subsequent analysis were performed as previously [17]. The cDNA complex was amplified using an iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) with the following primer sets: K-RAS, 5’-TGT TCA CAA AGG TTT TGT CTC C-3’ (forward) and 5’-CCT TAT AAT AGT TTC CAT TGC CTT G-3’ (reverse); CLDN1, 5’-TTT ACT CCT ATG CCG GCG AC-3’ (forward) and 5’-GAG GAT GCC AAC CAC CAT CA-3’ (reverse); CLDN7, 5’-AGT TAG GAG CCT TGA TGC CG-3’ (forward) and 5’-GCA CAG GGA GTA GGA TAC GC-3’ (reverse); RAB25, 5’-CCA TCA CCT CGG CGT ACT AT-3’ (forward) and 5’-TTT GTT ACC CAC GAG CAT GA-3’ (reverse); β-actin, 5’-AGA GCT ACG AGC TGC CTG AC-3’ (forward) and 5’- AGC ATC GTG TTG GCG TAC AG-3’ (reverse). The β-actin gene was used as a control for calculation of dCt value. The qRT-PCR data were analyzed using the 2-(ddCt) method.
2.5. Immunoblotting
Immunoblotting was performed as previously described [18]. E-cadherin antibody (610182) was purchased from BD Biosciences (San Jose, CA, USA). Antibodies for Rab25 (4314), Snail (3879), Twist (46702), Ras (3965), p-EGFR (3777) and EGFR (2085) were obtained from Cell Signaling Inc. (Danvers, MA, USA). Antibodies for β-actin (47778) claudin-1 (sc-166338) and claudin-7 (sc-17670) were purchased from Santa Cruz Biotechnology (Texas, CA, USA). K-Ras antibody (ab180772) was purchased form Abcam (Cambridge, UK). All primary antibodies were used at 1:1000 dilutions. Secondary antibodies for anti-rabbit and anti-mouse were from Thermo Fisher Scientific (1:3000, Waltham, MA, USA), while an anti-goat secondary antibody was purchased from Santa Cruz Biotechnology (1:3000 dilutions).
2.6. Immunofluorescence
Immunofluorescence was performed as previously described [19]. Briefly, antibodies of E-cadherin (610182, 1:500; BD Bioscience), Snail (sc-271977, 1:500; Santa Cruz Biotechnology), and claudin-7 (ab207300, 1:500; Abcam) were used at a dilution of 1:500. The cells were reacted with Cy2-conjugated goat anti-mouse IgG (111-223-003, 1:500; Jackson ImmunoResearch, PA, USA) and Cy3-conjugated goat anti-rabbit IgG (111-156-003, 1:500; Jackson ImmunoResearch, PA, USA). The nuclei of the cells were stained with 4’, 6’-diamidino-2-phenylindole dihydrochloride (62249, 1:1000, Thermo fisher Scientific, Waltham, MA, USA) at room temperature for 5 min. Fluorescence images were taken by confocal microscopy (LSM710; Carl Zeiss, Jena, Germany).
2.7. Wound healing assay
Wound healing assay was performed as previously reported [20]. The cells were cultured to confluence in 35 mm dish and transfected for 24 h. The cells were scraped with a micropipette tip. Photographic images were captured immediately after scraping and after 24 h, 36 h in the same locations. Wound closure rate was determined through the image J after 3 repeated experiments, and the average value was shown.
2.8. In vitro migration and invasion assay
In vitro migration assay was performed in triplicates using a 48-well chemotaxis chamber as described previously [17]. 1% FBS medium was added to the each well of the lower chamber, and type 1 collagen-coated 8 µm or 12 µm pore membranes (Neuro Probe, Gaithersburg, MD, USA) were added. The transfected cell suspension, the volume of 2×106 cells/well, was placed to each well of the upper chamber. After incubation for 6 h at 37°C, invaded cells were fixed and stained with Diff-Quik reagents (Dade Behring Inc., Newark, DE, USA). The average numbers of three random microscopic fields (×200) of invasion filters were counted in each experiment. In vitro invasion assay was used in triplicates using 48-well chemotaxis chamber as previously [21]. 1% FBS medium was added to each well of the lower chamber, and Matrigel-coated 8 µm or 12 µm membrane was added. The transfected cell suspension, the volume of 2×106 cells/well, was placed to each well of the upper chamber. After incubation for 16 ~ 18 h at 37°C, invaded cells were fixed and stained with Diff-Quik reagents (Dade Behring Inc., Newark, DE, USA). The average numbers of three random microscopic fields (×200) of invasion filters were counted in each experiment.
2.9. Three-dimensional (3D) Matrigel invasion assay
3D Matrigel invasion assay was performed as described previously [22–25]. Cancer cells were labeled with DiI (Thermo fisher Scientific, Waltham, MA, USA). The mixture was prepared by mixing of 20% type I collagen (Nitta Gelatin Inc, Cell matrix Type I-P, Japan) and Matrigel (Corning, Acton, MA, USA) and solidify on the in 3 µm pore size transwell inserts (Corning, Acton, MA, USA). 5×104 cells were mixed in 200 µl of medium supplemented with 0.2% FBS and plated on the gels. The 24-well plate filled with culture medium or serum-free medium. After 3 ~ 5 days, the embedded gel was sectioned without fixation, and the cells were analyzed by a fluorescence confocal microscopy. In these images, the distance of invaded cells was measured from five different positions and calculated by the ZEN blue edition program of Carl Zeiss Microscopy GmbH. The distance in µm was calculated as described previously [26].
2.10. Three-dimensional (3D) Matrigel culture
3D cultures were observed as described previously [18]. Total 3×104 cells/ml cells were suspended in 400 µL medium supplemented with 2% Matrigel and seeded over a layer of the 100% Matrigel in 8-well culture slide (#345108; Corning, Acton, MA, USA). Cells were grown for 7 days and media was changed every 2 days. Cells were monitored every day and examined using a light microscope.
2.11. Measurement of RAS activation using ELISA assay
The supernatants were removed and determined RAS activation using a RAS activation ELISA assay kit (#17–497, Millipore, Temecula, CA, USA) according to the manufacturer’s instructions. The level of activated RAS compared with the vector levels of activated RAS. The results represent triplicated experiments.
2.12. Ras-GTP pull down assay
The cell lysates were according to the manufacturer’s recommendation (Cytoskeleton Inc., #BK008, Denver, Co, USA). The lysates were incubated with Raf-RBD beads for 1 h at 4℃, and then eluted with 2X sample buffer. Samples were resolved by SDS-PAGE. After that, procedures were same immunoblotting. Antibodies for Pan-Ras (3395, Cell Signaling Inc.), K-Ras (sc-30, Santa Cruz Biotechnology), GST (sc-138, Santa Cruz Biotechnology) were used at 1:1000 dilutions.
2.13. Statistical analyses
Data are shown as the means ± standard deviation (s.d.). Statistical analysis was assessed with SigmaPlot software (SYSTAT Software, San Jose, CA) using the Student’s t-test. A P value less than 0.05 was reflected statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ns; no statistically significant difference). Differences among three or more groups were estimated by analysis of variance followed by Bonferroni multiple comparison tests.