Fig 1.
Increased UNC50 expression in HCC.
(A) Northern blotting analysis of UNC50 mRNA expression levels in 12 paired HCC tissues (T) and adjacent non-cancerous tissues (N); 28S and 18S bands were used as the references. (B) Real-time PCR analysis of UNC50 expression levels in 44 paired tissues. Relative mRNA expression levels are normalized to β-actin; log2-transformed fold changes of HCC tissues compared to the adjacent non-cancerous tissues were calculated by the comparative cycle threshold (ΔΔCt) method. The paired tissues are ordered from low to high ΔΔCt values. Cutoff values were set to ±1 to identify the significance of changes. (C) Western blotting analysis of UNC50 levels in another 12 paired HCC tissues; β-actin was used as the reference.
Fig 2.
Confirmation of microarray results in UNC50 overexpression and knockdown Hep3B cells.
(A) Western blotting confirmation of the effectiveness of UNC50 overexpression and knockdown; β-actin served as the reference. (B, C) Real-time PCR confirmation of seven regulated genes in response to UNC50 (B) knockdown or (C) overexpression; expression levels were normalized against β-actin. **: p<0.01.
Table 1.
List of main regulated genes downstream of the EGFR pathway in UNC50 knockdown Hep3B cells.
Fig 3.
Western blotting confirmation that UNC50 affects cellular EGFR pathway activity.
Cells (105 from each cell type) were seeded to 24-well plates in DMEM with 10% FBS (Complete medium). After adherence, cells were pre-treated with serum free DMEM for 24 hours before being stimulated with either flesh serum free DMEM, complete medium, or complete medium with 1ng/ml EGF for 8 hours. Lysates were then collected and analyzed for pEGFR, EGFR, β-actin, cyclin D1 and UNC50 by western blot. SF = serum-free DMEM.
Fig 4.
UNC50 prompts EGF-induced cell cycle entry and proliferation.
(A) Flow cytometry analysis of cells stimulated with complete medium (10% FBS), complete medium with 1 ng/ml EGF (EGF) added for another 24 hours, or 10 μmol/ml erlotinib (Erlotinib) after EGF treatment. (B) MTT evaluation of cell proliferation status. Top: Cells were cultured in complete medium without change of medium; cell vitality was analyzed every 24 hours. Bottom: Cells were cultured in complete medium with 1 ng/ml EGF and replaced with fresh medium every 12 hours to sustain EGF levels. Cell vitality was analyzed every 24 hours. *: p<0.05, **: p<0.01.
Fig 5.
Flow cytometry and immunofluorescence analyses of EGFR distribution.
(A, B) Cells not treated with 0.05% Triton X-100 before antibody staining, (C, D) Cells permeabilized by 0.05% Triton X-100 before antibody staining. Isotype rabbit immunoglobulin G (IgG) was used as the negative control. (E) Cells were stained for EGFR with Alexa 488-conjugated EGFR antibody and visualized under a fluorescence microscope at 488 nm excitation light. Scale bar is 10 μm.
Table 2.
Gmh1p interaction partners in yeast.
Fig 6.
Distribution of recombinant UNC50 protein.
Hep3B cells at approximately 40% confluence were transfected with 1 μg pEGFP-N1-UNC50. After 48-hour transfection, cells were fixed in 4% paraformaldehyde and stained for Golgi apparatus, endoplasmic reticulum, and early endosome markers AKR7A2, calnexin 1, and early endosomal autoantigen 1 (EEA1), respectively. Cells were examined under a fluorescence microscope at corresponding excitation light. Scale bar is 10 μm.