Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production

doi: 10.2183/pjab.87.563

Figure Lengend Snippet: Biological function of NEDD4 in human normal fibroblasts. (A) NEDD4 was expressed in normal human skin, normal human dermal fibroblasts (NHDF), human keratinocytes (NHEK) and keloid fibroblast, respectively. β -actin ( ACTB ) was used for quantitative normalization. (B) Cell viability assay showing that NEDD4 promoted cell proliferation. MTT assay of NHDF cells that were transfected with NEDD4 expression vector or the control mock vector. ABS on Y-axis means absorbance at 490 nm with that at 630 nm as a reference measured with a microplate reader. These experiments were carried out in triplicate (* P = 0.0001). (C) NEDD4 over-expressing NIH3T3 cells promoted cell invasion by Matrigel invasion assay. Invasion index means the average cell number of migration, counted by microscopic observation. These experiments were carried out in triplicate (** P = 0.0235). Error bars represent mean ± s.d. (D, E) NEDD4 introduced NIH3T3 cells were multi-layered after cells reached 100% confluent (D) and formed colonies (E), whereas these were not observed in control mock cells.

Article Snippet: Normal Human Dermal Fibroblast (NHDF) and normal Human Epithelial Keratinocyte (NHEK) were purchased from Lonza (Walkersvill, MD).

Techniques: Viability Assay, MTT Assay, Transfection, Expressing, Plasmid Preparation, Control, Invasion Assay, Migration

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production

doi: 10.2183/pjab.87.563

Figure Lengend Snippet: NEDD4 increased cytoplasmic β-catenin and upregulated β-catenin transcriptional activity. (A) Up-regulation of β-catenin in keloid fibroblasts. Endogenous β-catenin was detected by western blot analysis using total cell lysates. NHDF; normal human dermal fibroblast, NHEK; normal human epithelial keratinocyte, short; short exposure, long; long exposure. (B) Phospho-GSK3β and β-catenin were up-regulated in the cells that NEDD4 was transiently introduced. The cells were incubated for 48 h or 72 h after the transfection. Depletion of NEDD4 in keloid fibroblasts decreased the protein level of β-catenin. The cells were incubated for 48 h after the transfection of siRNA duplex. (C) Immunocytochemical staining detected β-catenin mainly at plasma membrane in the cells in which NEDD4 was not introduced while β-catenin was translocated to the cytoplasm in NEDD4 over-expressing cells. Bar = 12 µm. (D) Transcriptional changes of the genes regulated in the β-catenin/TCF were analyzed by semi-quantitative RT-PCR. Cells were incubated for 72 h (for transiently expressing) or 96 h (for knockdown). β -actin ( ACTB ) was used to quantify cDNA contents.

Article Snippet: Normal Human Dermal Fibroblast (NHDF) and normal Human Epithelial Keratinocyte (NHEK) were purchased from Lonza (Walkersvill, MD).

Techniques: Activity Assay, Western Blot, Incubation, Transfection, Staining, Clinical Proteomics, Membrane, Expressing, Quantitative RT-PCR, Knockdown