Fig 2: Chemicals treatment of PANC-1(a) PANC-1 cells overexpressing INPP4B were incubated in RPMI containing CHX and lysed for western blotting detection of Ecad and INPP4B. Mock infected and uninfected cells were parallelly treated with CHX and served as controls. (b, c) PANC-1 cells infected by Ad-INPP4B were treated with RG108 and probed for Ecad and INPP4B by western blot and RT-qPCR. DMSO-treated and untreated cells served as controls. The quantitative results of western blot and PCR were analyzed basing on gray-level ratio of target band and 2-??Ct method. GAPDH was used as housekeeping gene for all data normalization. (d) Sequenom MassARRAY platform was used for the quantitative methylation analysis. The colors of each circle represent the methylation level of each corresponding CpG unit. Quantitative methylation analysis results are shown in a color scale: yellow (0% methylation), green (50% methylation), and dark blue (100% methylation). The white circles represent the missing data at a given CpG site. (e) The significantly changed methylation levels of certain CpG units were shown. (f) Mean methylation percentage of total CpG sites evaluated in the control group and RG108 group was compared. Results show mean ± SD (n = 3). N/S and * indicate P > 0.05 and P < 0.05.

Fig 3: INPP4B expression in four PDAC cell lines and its impact on EMT marker in PANC-1 and aspc-1. Relative INPP4B expression level in four pancreatic cancer cell lines was quantified by western blot (a) and RT-qPCR (b) and listed as follows : AsPC-1> BxPC-3> SW1990> PANC-1 (All P < 0.05). The PANC-1 with relatively minimal INPP4B level was chosen for overexpression assay, and AsPC-1 with the highest INPP4B level among was selected for knockdown use. INPP4B overexpression induced upregulation of Ecad and Vim with downregulated Ncad in PANC-1 compared to that of the control in protein (c) and transcript levels (d). Knockdown of INPP4B in AsPC-1 led to reduction of Ecad and Vim with increased Ncad compared to control group (e, f). These results were further substantiated by immunofluorescence analysis for Ecad (g, top panel), Ncad (middle panel) and Vim (bottom panel) expression. Scale bar in images = 20 mm. The quantitative results of western blot and PCR were analyzed basing on gray-level ratio of target band and 2-??Ct method. GAPDH was used as housekeeping gene for all data normalization. Results show mean ± SD (n = 3). N/S and * indicate P> 0.05 and P < 0.05. The boxes and arrowheads in merge images indicate regions of interest.

Fig 4: Expression of INPP4B and ecad in pancreatic tumor specimens detected by immunohistochemistry. Shown were representative photomicrographs (×400) of negatively (a, b) and positively stained specimens (c-f). Epithelial cancer cells with more cytoplasmic expression of Ecad than membranous expression (e), and cells with only membranous loss of Ecad (f) were detected. Relevance of target protein expression to tumor grade (g), lymph node metastasis status (h) and TNM stage (i) of tissues was expounded by Mann-Whitney U test. N0 indicates no lymph node metastasis. N1 indicates regional lymph node metastasis. Scale bar in images = 100 mm. Results show mean ± SD (n = 3). N/S and * indicate P > 0.05 and P < 0.05.

Fig 5: Down regulation of TET2 and TET3 in the TGF-ß1-induced EMT-like process in A375 cells(A) Representative morphologic changes of A375 cells cultured with or without TGF-ß1 (5 ng/ml) for 3 days were shown on the left, Scale bar = 50 µm, the percentage of spindle cells were shown on the right; (B) The effect of TGF-ß1 on cell growth was evaluated by measuring the absorbance of OD492 after staining with MTT; (C) and (D) Real-time RT-PCR analysis of the relative expression levels of CDH1, CDH2 and TET1/2/3 mRNAs in A375 cells stimulated with 5 ng/ml TGF-ß1 for the indicated times. The data are displayed as the fold change in the expression levels in untreated cells; the levels were normalized to those of GAPDH, and the error bars represent the mean ± SD of triplicate experiments (Student's t-test, *p < 0.05, **p < 0.01); (E) Expression of E-cadherin, N-cadherin and TET1/2/3 in A375 cells cultured with 5 ng/ml TGF-ß1 for the indicated times was examined by immunoblotting. GAPDH was used to show that equal amounts of proteins were loaded on the gel; (F) Immunoblotting analysis of endogenous TET2 and TET3 expression levels in A375 cells and SK-MEL-1 cells that were treated with vehicle (0), the TGF-ß type I receptor inhibitor LY9702161 or that were stimulated with TGF-ß1 for 3 days, GAPDH was used to show that equal amounts of proteins were loaded on the gel; (G) The 5hmC levels of the genome of the cells treated with or without TGF-ß were detected by dot-blotting.