Supplementary MaterialsSupplementary figure 1 41388_2018_403_MOESM1_ESM. cancer affected person survival generated with the Tumor Genome Atlas (TCGA). low (FPKM??6) and great (FPKM? ?6) appearance group contained 54 and 122 individual examples, respectively. c Representative pictures displaying MUC20 overexpression in pancreatic tumour tissue weighed against the adjacent non-tumour tissues by immunohistochemistry (IHC) of tissues microarray (US Biomax, Inc). Size bar signifies 50?m. d Scatter story graph represents the MUC20 expression rating in tumour and non-tumour servings from the pancreas. MUC20 appearance was have scored by multiplication of strength (0C3) and positive region (1C3). Data are shown as mean (analysed by real-time RT-PCR in PDAC cell lines, as indicated. b The proteins degrees of MUC20 analysed by American blotting in PDAC cell lines. c Western blots showing MUC20 knockdown with two impartial siRNAs (si-MUC20-1 and si-MUC20-2) in HPAC and HPAF-II cells. d MUC20 knockdown inhibited viability in HPAC and HPAF-II cells analysed by MTT assays. *was upregulated by serum deprivation in HPAC and HPAF-II cells (Supplementary Fig. S3A). Serum deprivation increased the activity of phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 (Supplementary Fig. S3B). Inhibition of p-JNK activity using SP600125 could suppress MUC20 expression induced by serum deprivation (Supplementary Fig. S3C), suggesting that this p-JNK signalling pathway is usually involved in the MUC20 induction by serum deprivation. These results suggest that MUC20 expression can be induced by tumour microenvironmental factors in PDAC cells, which include CFPAC-1, Capan-2, HPAC, and HPAF-II cell lines. Open in a separate windows Fig. 4 MUC20 is usually up-regulated in serum-deprived, hypoxic, and acidic microenvironment. a MUC20 was induced by serum deprivation (0% FBS). b PF-4136309 MUC20 was induced by hypoxia (1% oxygen). c MUC20 was induced by acidic condition (pH 6.5). PDAC cells were treated with these different microenvironmental factors for 24?h. The expression of MUC20 was analysed PF-4136309 by western blotting. -actin was used as an internal control. Statistical results for MUC20 signals are shown. Data are presented as mean (sense, 5-CGTGCGTGACATTAAGGAGA-3 and anti-sense, 5-GAAGGAAGGCTGGAAGAGTG-3; sense, 5-AACTCCACGCCCACGCGCCT-3 and anti-sense, 5-GGAAGCACACAGATGGGTG-3; sense, 5-ATGATGTCCACGGAAGAGGAGA-3 and anti-sense, 5-CACTCGTAATAGGCCATCATAGTTGA -3. Transfection and plasmid construction For transient MUC20 knockdown, two impartial siRNAs and non-targeting siRNA (Dharmacon, ThermoFisher Scientific, MA, USA) were used to transfect PDAC cells by Lipofectamine RNAiMAX (Invitrogen) with a final concentration of 10?nM for 3 days. For stable MUC20 knockdown and its control cells, sh-MUC20/pLKO.1 plasmid and pLKO.1 vector (RNAi Core, Academia Sinica, Taiwan) were used in lentivirus-based PF-4136309 infection system, respectively, and selected with 2?g/ml puromycin (Sigma. St. Louis, MO, USA). MUC20 overexpression and its mock control cells were established by transfection of MUC20/pcDNA3.1?A plasmid or pcDNA3.1?A vector, respectively, using Lipofectamine 3000 (Invitrogen) according to the manufacturers protocol. Human wild-type (NCBI Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001282506.1″,”term_id”:”541444091″,”term_text”:”NM_001282506.1″NM_001282506.1) and truncated were cloned using PCR kit (Invitrogen). The sense primer was 5-AAGCTTATGGGCTGTCTCTGGGGTCT-3. Antisense primer for wild-type was 5-GGATCCTTAGCCTCTCCTGACACGCA-3. Antisense primer for truncated was 5-GGATCCTTATGCACTCACGTCTGTGGTC-3. The PCR products were cloned into pcDNA3.1/myc-His (Invitrogen) to generate the MUC20/pcDNA3.1A plasmid. The MUC20 was confirmed by DNA sequencing. AKT/PCIS2 plasmid and its control vector, PCIS2, Plau were gifts from Dr. Michael J. Quon (University of Maryland School of Medicine, Division of Endocrinology, USA). Reagents and Antibodies MUC20 antibody was prepared as described inside our previous research [24]. Antibody against -actin (A5441) was extracted from Sigma. Antibodies against MET (GTX100637), AKT (GTX121937),.