Fig 1: Immunohistochemical staining for KIAA1199, E-cadherin, N-cadherin and vimentin. (A). A1, A2: Positive KIAA1199 expression in CCA tissue. E1, E2: Negative KIAA1199expression in adjacent tissue. B1, B2: Negative E-cadherin expression in CCA tissue. F1, F2: Positive E-cadherin expression in adjacent tissue. C1, C2: Positive N-cadherin expression in CCA tissue. G1, G2: Positive Vimentin expression in CCA tissue. H1, H2: Negative Vimentin expression in adjacent tissue. (scale bar, 50 µm; magnification: ×200, ×400) (B) Western blot analysis of EMT signaling molecules (N-cadherin, E-cadherin and Vimentin) in KIAA1199 silenced Hucct1 cell line. (C) Western blot analysis of EMT signaling molecules ((N-cadherin, E-cadherin and Vimentin) in KIAA1199 overexpressed QBC939 cell line. Representative of three independent experiments.
Fig 2: (A) Immunofluorescence localization of KIAA1199 and CK-18 in cholangiocarcinoma tissue (red, KIAA1199; green, CK-18; blue, DAPI). (B) Immunofluorescence localization of KIAA1199 and Phalloidin in Hucct-1 cell line (red, KIAA1199; green, Phalloidin; blue, DAPI). (C) Microscopic determination of KIAA1199 cellular localization using QBC939 cells transfected with green fluorescent protein (GFP), KIAA1199-GFP chimeric cDNAs. (D) KIAA1199 had signal peptides. (E) KIAA1199 has 602 amino acids, all of which are extracellular, and there is no transmembrane domain (TMD). (F) Western blot analysis was performed to detect KIAA1199 in cell lysates and conditioned culturing medium from Hucct-1 and Hucct-1 knockdown KIAA1199 (siKIAA1199).
Fig 3: (A) Kaplan-Meier analysis of overall survival (OS) and disease-free survival (DFS) in 177 patients with CCA according to KIAA1199 staining. (B, C) Univariate and multivariate analyses of factors associated with survival and recurrence.
Fig 4: KIAA1199 regulates proliferation and invasion in CCA cell lines. (A, B) The relative protein and mRNA expression of four small interfering RNA in siKIAA1199-transfected cells compared with control and parental cells. (F, G) Overexpression of KIAA1199 in QBC939 cells with lentivirus infection was verified by western blotting and qPCR. Proliferation of Hucct-1 cells was detected with CCK-8 after silencing KIAA1199 in Hucct-1 cells (C) and overexpressing KIAA1199 in QBC939 cells (H) in normal medium with 10% FBS. Wound healing assay was applied to evaluate migration of Hucct-1 (D) and QBC939 (I). 24 h after a scratch in the cell monolayer, the wound size was measured again. Migration of Hucct-1 and QBC939 cells was assessed with transwell assay (E, J). After KIAA1199 knockdown and overexpression, cells were seeded in the upper transwell chamber and incubated for 24 h, with FBS in the lower chamber. (original magnification: ×200; scale bar, 20 µm). Data, mean ± S.D., and representative of three independent experiments.
Fig 5: The expression of KIAA1199 and TGF-ß-PI3K-Akt pathway-associated proteins by western blot analyses. (A) Western blot analysis of KIAA1199 and TGF-ß-PI3K-Akt pathway-associated proteins in KIAA1199 silenced Hucct1 cell line. (B) Western blot analysis of KIAA1199 and TGF-ß-PI3K-Akt pathway-associated proteins in KIAA1199 overexpressed QBC939 cell line. (C) QBC939 were pretreated with TGF-ß inhibitor (SB431542, 5 µM) for 2 h and transwell migration assay was performed in the absence or presence of KIAA1199 conditioned medium (CM). (D) QBC939 were pretreated with PI3K inhibitor (LY294002, 6µM) for 2 h and trans-well migration assay was performed in the absence or presence of KIAA1199 conditioned medium (CM). (E) KIAA1199-mediated EMT may occur through a non-Smad pathway. At least three independent experiments were preformed, data presented as mean ± SD, *, ** and *** represented P<0.05, P= 0.01 and 0.001 by Student's t-test, between the indicated group.
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