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  • br Knockdown of CLDN gene by siRNA


    Knockdown of CLDN7 gene by siRNA
    The A 61603 were seeded in 6 or 10-cm Petri dishes (Corning Inc., Corning, NY, USA or TPP Techno Plastic Products, Trasadingen, Switzerland). Following overnight incubation, siRNA specific for CLDN7 or negative control siRNA (Sigma-Aldrich, St. Louis, MO, USA) transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to manufacturer's instructions and further incubated for 24 or 48 h.
    Reverse transcription-polymerase chain reaction (RT-PCR)
    Total RNA was extracted using an RNeasy Plus Mini kit (Qiagen GmbH, Hilden, Germany). cDNA was synthesized from 500 ng of total RNA using a PrimeScript RT Reagent kit (Takara Bio, Shiga, Japan) and the GeneAmp PCR System 9700 (Thermo Fisher Scien-tific). For RT-PCR detection of CLDN7 and 18S rRNA, 100 ng of cDNA was amplified using TaqMan Universal Master Mix II, no UNG (Thermo Fisher Scientific) with the 7300 Real-Time PCR System (Thermo Fisher Scientific). The PCR conditions consisted of an initial denaturation step (95 C for 10 min) followed by 40 cycles (95 C for 15 s and 60 C for 1 min). The TaqMan primers for the CLDN7 (Assay ID: Hs00600772_m1) and 18S ribosomal RNA (Assay ID: Hs99999901_s1) genes were purchased from Thermo Fisher Scientific. Relative expression was calculated using the delta-delta Ct method [33].
    Cell cycle analysis
    The MIA PaCa-2-A cells were untreated or transfected with CLDN7-specific siRNA or control siRNA. After 24 h incubation, untreated or siRNA-transfected cells were synchronized by 48 h serum starvation. The cells were then detached by trypsin-EDTA traetment and further incubated in the complete me-dium without siRNA.The cells were harvested immediately af-ter the serum starvation and after following 24 h incubation. They were fixed with 70% (v/v) ethanol and stored at 20 C until use. Following centrifugation, the cell pellet was washed twice with PBS, re-suspended in PI/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA), and incubated at room temperature for 15 min. The cells were then analyzed using a MACSQuant Analyzer (Miltenyi Biotec K.K., Bergisch Gladbach, Germany). 
    Statistical analysis
    Data are expressed as the means values ± standard deviation. A non-paired t-test and repeated measures analysis of variance were used for statistical analyses. All P-values were considered to indi-cate statistically significant differences when the associated prob-ability was <0.05.
    Isolation of clonal cells with high and low malignancy phenotypes from MIA PaCa-2 cells
    Under a phase contrast microscope, the MIA PaCa-2 cells consisted of a mixture of large epithelial-like cells that firmly attached to the plastic dish and small non-epithelial-like cells that were loosely attached to the plastic dish (Fig. 1A). Following single cell cloning by the repeated limited dilution method, clonal cell populations with each morphological characteristic could be isolated; 7 epithelial-like clones and 9 non-epithelial-like clones were obtained. A representative population of clonal cells exhibiting an epithelial-like morphology was designated as the MIA PaCa-2-A cells, while the population of cells exhibiting a non-epithelial-like morphology was designated as the MIA PaCa-2-R cells (Fig. 1A). The letter ‘A’ in MIA PaCa-2-A indicates ‘ad-hesive’ and ‘R’ in MIA Paca-2-R indicates ‘round’. The images in the upper panels in Fig. 1A show the MIA PaCa-2-A and MIA PaCa-2-R cells directly after establishment, and the images on the lower panels show each cell population maintained for >4 years, showing that each clonal population maintained its morpholog-ical characteristics and did not exhibit any change in morphology even after long-term culture. DNA STR analysis demonstrated that the MIA PaCa-2-A and MIA PaCa-2-R cells had the same pattern of DNA STR identical to that of the parental MIA PaCa-2 cells (Supplement Table 1 and Fig. 1B), indicating that the MIA PaCa-2-A and MIA PaCa-2-R cells were derived from the MIA PaCa-2 cells and were not contaminated cells by other cell lines. The activation of Erk1/2, Akt, NF-kB and STAT3 pathways is closely associated with the morphogenesis, proliferation, survival and drug resistance of malignant cells [34e36]. A higher expression of p-NF-kB was observed in the MIA PaCa-2-A cells compared with the MIA PaCa-2-R cells, while p-STAT3 expression was observed in the MIA PaCa-2-R cells, but not in the MIA PaCa-2-A cells (Fig. 1C). The expression of p-Erk1/2 was similar be-tween the MIA PaCa-2-A and MIA PaCa-2-R cells, and the expression of p-Akt was not seen in both the MIA PaCa-2-A and MIA PaCa-2-R cells.