grp78 overexpression plasmid  (GenScript corporation)

Journal: Journal of cellular biochemistry

Article Title: Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

doi: 10.1002/jcb.29903

Figure Lengend Snippet: FIGURE 1 GGA induces Grp78 expression in hPDLCs without affecting proliferation. (A) hPDLCs were stimulated with 200 μM GGA for the indicated intervals. The total RNA was isolated and analyzed to evaluate Grp78 mRNA expression using quantitative real‐time PCR. Data are represented as fold increases in the expression relative to that of GAPDH (n = 3). (B) After stimulation by GGA for the indicated intervals, whole‐cell lysates prepared from hPDLCs were subjected to immunoblot analysis with an anti‐Grp78 antibody. β‐actin was used as a control. Representative western blot analysis of total Grp78 is shown in the upper panel. Quantification of total Grp78 protein levels relative to β‐actin was performed using ImageJ (lower panel) (n = 3). (C) Diagram of the protocol for hPDLCs treatment: unstimulated cells, GGA, rM180, or GGA + rM180. Functional analysis with hPDLCs was performed at the indicated time points after the first medium exchange following 18 h of culture with media alone, with GGA, or rM180 (10 μg/ml). (D) WST‐8 assay was performed for the indicated intervals. Proliferation is expressed as the absorbance measured at 450–655 nm (n = 5). Data are represented as mean ± SD. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GGA, geranylgeranylacetone; Grp78, glucose‐regulated protein 78; hPDLC, human periodontal ligament cell; mRNA, messenger RNA; N.S., not significant; PCR, polymerase chain reaction. *p < .05; **p < .01; ***p < .001

Article Snippet: At approximately 60%–70% confluence, the cells were transfected with 2 μg of the Grp78‐ overexpression plasmid (pcDNA3.1 (+) Grp78/Bip, RRID: Addgene_32701; Addgene) using 2 μl of Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Control, Functional Assay, Polymerase Chain Reaction

Journal: Journal of cellular biochemistry

Article Title: Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

doi: 10.1002/jcb.29903

Figure Lengend Snippet: FIGURE 7 Proposed mechanism of wound healing in response to combined treatment with GGA and rM180 amelogenin in hPDLCs. GGA increases Grp78 expression and enhances the migration of hPDLCs. The increased Grp78 expression triggers the production of Angptl4 and Areg, which are involved in angiogenesis and wound healing, via the activation of HIF‐1α and PPARδ and the phosphorylation of CREB and PKA. Supplementation with rM180 amelogenin not only strongly enhances the migratory capacity of hPDLCs, but also accelerates their angiogenic activity owing to the upregulation of IL‐8, MCP‐1, and IL‐6. Angptl4, angiopoietin‐ like 4; Areg, amphiregulin; CREB, cAMP response element‐binding protein; GGA, geranylgeranylacetone; HIF‐1α, hypoxia‐inducible factor 1α; hPDLC, human periodontal ligament cell; IL‐8, interleukin‐8; MCP‐1, monocyte chemotactic protein 1; PKA, protein kinase A; PPARδ, peroxisome proliferator‐activated receptor δ

Article Snippet: At approximately 60%–70% confluence, the cells were transfected with 2 μg of the Grp78‐ overexpression plasmid (pcDNA3.1 (+) Grp78/Bip, RRID: Addgene_32701; Addgene) using 2 μl of Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Migration, Activation Assay, Phospho-proteomics, Activity Assay, Binding Assay