pegfp shp2  (Addgene inc)

Journal: BMC Cancer

Article Title: Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis

doi: 10.1186/1471-2407-14-442

Figure Lengend Snippet: Upregulation of SHP2 expression correlates with the migratory and invasive ability of oral cancer cells. (A) Oral tumors and histologically normal oral mucosa adjacent to the tumors were stained with anti-SHP2 antibody. The IHC semi-quantitative score was derived by two independent pathologies, multiplying the staining intensity by the percent of tumor cells stained. IHC scores for each core of a specimen were averaged (n = 19) and statistically analyzed. (B) cDNA from paired oral tumor samples were subjected to RT-PCR (n = 18). Relative expression of SHP2 transcript to internal control gene, GAPDH was calculated as described in Materials and Methods. (C) Cell proliferation was performed by MTT assay. Cells were counted at 570 nm wavelength and the relative absorbance was represented as mean ± SD from at least four independent experiments. (D) Cells were seeded onto the transwell chamber coated with matrigel as described in Methods. Images are representative of cells adhering to the lower chamber after the invasive process. Cells were stained with crystal violet solution, and images were taken by photography (Upper panel). Invading cells per file on the lower chamber were counted. The data are expressed as mean ± SD from three independent experiments; P < 0.05 . (Lower panel) (E) An increased SHP2 transcript level was associated with higher invasive ability of HSC3 cells. The expression of SHP2 for HSC3-Inv4 and HSC3-Inv8 was normalized to HSC3 parental cells.

Article Snippet: The pEGFP-SHP2 WT and C/S mutant were engineered by inserting a coding region into the SalI and BamHI sites of pEGFP vector (Stratagene).

Techniques: Expressing, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, MTT Assay

Journal: BMC Cancer

Article Title: Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis

doi: 10.1186/1471-2407-14-442

Figure Lengend Snippet: SHP2 depletion or catalytic deficiency mutant inhibits migration and invasion of oral cancer cells. (A) Cells transfected with SHP2 si-RNA (si-SHP2#1 or si-SHP2#2) or Negative control (si-NC) were seeded onto the transwell chamber coated with or without matrigel as described in Materials and Methods. Cells adhering to the lower chamber after the migration or invasive process were stained with crystal violet solution, and images were taken under bright-field microscopy at 40×. An obvious decrease in migration (Upper panel) and invasion (middle panel) ability was noted in HSC3 cells transfected with SHP2 si-RNA (si-SHP2#1 or si-SHP2#2) compared to Negative control (si-NC). Western blot shows the expression of SHP2 in HSC3 cells transfected with SHP2 si-RNA or Negative control (Lower panel). (B) Effect of SHP2 knockdown on invasion of HSC3-Inv4 and HSC3-Inv8 cells (Upper panel, left and right, respevtively). The quantitative data are expressed as mean ± SD from three independent experiments; *, P < 0.05 (Middle panel). Western blot shows the expression level of SHP2 in HSC3-Inv4 and HSC3-Inv8 cells transfected with SHP2 si-RNA or Negative control (Lower panel, left and right, respectively). (C) A dramatic decrease in migration (Left panel) and invasion ability (Middle panel) was observed in HSC3 cells transfected with SHP2 C459S mutant (SHP2C/S) compared to the SHP2 wild type (SHP2WT). Evaluation on SHP2 activity of the cells transfected with indicated constructs. Experiments were done in triplicate at least, and values are indicated as mean ± SD. *, P < 0.05 (Right upper panel). Western blot shows the expression level of transfected flag-SHP2 proteins (Right lower panel).

Article Snippet: The pEGFP-SHP2 WT and C/S mutant were engineered by inserting a coding region into the SalI and BamHI sites of pEGFP vector (Stratagene).

Techniques: Mutagenesis, Migration, Transfection, Negative Control, Staining, Microscopy, Western Blot, Expressing, Activity Assay, Construct

Journal: BMC Cancer

Article Title: Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis

doi: 10.1186/1471-2407-14-442

Figure Lengend Snippet: Characteristics of highly invasive clone, HSC3-Inv4 derived from parental HSC3 cells. (A) Bright file microscopy images of HSC3 parental and HSC3 Inv 4 (20×, Upper panels). Cells were stained with E-cadherin and images were taken under fluorescence at 60× (Lower panels). (B) Expressions of E-cadherin and vimentin were analyzed by Western blot with indicated antibodies; GAPDH as a loading control. (C) Increased Snail (Upper panel) and Twist1 (Middle panel) transcript levels were observed in HSC3-Inv4 and HSC3-Inv8 compared to HSC3 parental cells. Experiments were done at least in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with the adjacent normal in each case. Western blot shows the expression level of Snail and Twist1 in HSC3-parental, HSC3-Inv4 and HSC3-Inv8 cells (Lower panel). (D) Status of MMP-2 secretion on highly invasive clones. Medium collected from HSC3 parental, HSC-Inv4 and HSC3-Inv8 cells were subjected to MMP-2 secretion analysis. Significantly increased amounts of MMP-2 were seen in selected sub-cell lines compared to parental cells. (E) SHP2 depletion resulted in decreased MMP-2 secretion in HSC3 parental, HSC3-Inv4 and HSC3-Inv8 cells.

Article Snippet: The pEGFP-SHP2 WT and C/S mutant were engineered by inserting a coding region into the SalI and BamHI sites of pEGFP vector (Stratagene).

Techniques: Derivative Assay, Microscopy, Staining, Fluorescence, Western Blot, Expressing, Clone Assay

Journal: BMC Cancer

Article Title: Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis

doi: 10.1186/1471-2407-14-442

Figure Lengend Snippet: SHP2 acts on Snail/Twist1 through negatively regulating ERK1/2 activity. (A) SHP2 forms a complex with ERK1/2. Total cell lysates were prepared, and SHP2 was immunoprecipitated from HSC3 cells expressing EGFP-tagged SHP2 wild type or catalytic-defective SHP2 (SHP2C/S). SHP2 in association with active ERK1/2 in these cells was detected by SDS-PAGE and immunoblotting with anti-phospho-ERK1/2, ERK1/2, SHP2 and GFP. (B) Nuclear localization of phospho-ERK1/2 is enriched in HSC3-Inv4 and HSC3-Inv 8 compared to HSC3 parental cells. (C) Treatment of ERK inhibitor with indicated concentration for 6 hours significantly reduced Snail or Twist1 mRNA expression in HSC3 parental and HSC3-Inv8 cells. (D) SHP2 depletion significantly increased Snail orTwist1 mRNA expression in HSC3 parental and HSC3-Inv8 cells (Upper panel and lower panel, respectively.). Experiments were done in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with adjacent normal in each case. (E) Knockdown of SHP2 increases both cytosol and nuclear localization of phospho-ERK1/2 in oral cancer cells. Poly ADP-ribose polymerase (PARP) was used as a nuclear marker.

Article Snippet: The pEGFP-SHP2 WT and C/S mutant were engineered by inserting a coding region into the SalI and BamHI sites of pEGFP vector (Stratagene).

Techniques: Activity Assay, Immunoprecipitation, Expressing, SDS Page, Western Blot, Concentration Assay, Marker

Journal: BMC Cancer

Article Title: Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis

doi: 10.1186/1471-2407-14-442

Figure Lengend Snippet: SHP2 promotes lung metastasis. SHP2 si-RNA delivered via tail vein injection dramatically reduced the metastatic capacity of HSC3 cells. Representative images showing H&E staining of lung tissues were taken under bright-field at 200× using a scanning microscope (Upper panel). Black lines delineate tumor tissue (T). Quantitative metastasis index was indicated as mean ± SD. *, P < 0.05 compared with the control group, HSC3 cells (Lower panel).

Article Snippet: The pEGFP-SHP2 WT and C/S mutant were engineered by inserting a coding region into the SalI and BamHI sites of pEGFP vector (Stratagene).

Techniques: Injection, Staining, Microscopy