Journal: Oncogene

Article Title: Repression of the MSP/MST-1 gene contributes to the antiapoptotic gain of function of mutant p53.

doi: 10.1038/sj.onc.1209061

Figure Lengend Snippet: Figure 1 Mutant p53 downregulates MSP expression. (a) Single-cell clones derived from H1299 cells either expressing mutant p53 (R175H) or an empty vector (pCMV-neo-Bam) were seeded in six-well plates. After 24 h, cells were harvested and total RNA was extracted. Equal amounts of RNA from several clones were pooled together, and 2 mg of each RNA pool was used as a template for reverse transcription. Real-time PCR was performed with primers specific for either MSP or GAPDH mRNA. Relative MSP mRNA levels were calculated by normalization of the MSP value for the amount of GAPDH transcripts in the same sample; results are shown in arbitrary units. (b) H1299 cells were transfected with either p53R175H or p53R273H expression plasmids or empty vector (control). Following 2 weeks of drug selection, single-cell clones were isolated. Equal amounts of total RNA, obtained from individual clones expressing either p53R175H or p53R273H, were pooled together (separately for each p53 mutant) before cDNA synthesis. cDNA was subsequently prepared and RNA levels of MSP and GAPDH were determined by semiquantitative RT–PCR. (c) H1299 cells were seeded in 24-well culture dishes. Each well was transfected with 0.2 mg of MSP-luciferase reporter plasmid DNA together with 100 ng of either pCMV-neo-Bam vector control (cont) or mutant p53R175H expression plasmid. Each plasmid combination was transfected into six identical wells. Luciferase activity was assayed 48 h post-transfection, and is shown in arbitrary machine units. The average and standard error are shown. (d) A total of, 100 000 H1299, SKBR3 and SW480 cells were seeded in six-well plates. H1299 cells were transfected with either 0.5 mg of a control plasmid or a plasmid expressing mutant p53 (R175H). The cells were collected 48 h later, and subjected to SDS–PAGE followed by Western blot analysis with p53-specific antibodies (see Materials and methods).

Article Snippet: Protein sample buffer was added to the medium concentrates, which were then resolved by SDS–PAGE on a 10% polyacrylamide gel and probed with monoclonal anti-MSP antibodies (AF352, R&D systems), followed by secondary goat anti-mouse HRP-conjugated antibodies (Jackson).

Techniques: Mutagenesis, Expressing, Clone Assay, Derivative Assay, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Control, Selection, Isolation, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, SDS Page, Western Blot

Journal: Oncogene

Article Title: Repression of the MSP/MST-1 gene contributes to the antiapoptotic gain of function of mutant p53.

doi: 10.1038/sj.onc.1209061

Figure Lengend Snippet: Figure 2 Mutant p53 associates with the MSP promoter in vivo. Extracts from H1299 cells stably transfected with either a vector control (p53 null) or mutant p53 R175H (mp53), as well as H1299 cells transiently transfected with an expression plasmid encoding wt p53 (wtp53), were subjected to ChIP using anti-p53 polyclonal antibodies, as described in Materials and methods. The presence of MSP genomic sequences was subsequently detected by performing PCR reactions on the immunoprecipitated genomic DNA. The no antibody (no AB) reaction was performed with material from control immunoprecipitations performed with the chromatin of mutant p53-transfected cells, from which the 0.1% input sample was also derived.

Article Snippet: Protein sample buffer was added to the medium concentrates, which were then resolved by SDS–PAGE on a 10% polyacrylamide gel and probed with monoclonal anti-MSP antibodies (AF352, R&D systems), followed by secondary goat anti-mouse HRP-conjugated antibodies (Jackson).

Techniques: Mutagenesis, In Vivo, Stable Transfection, Transfection, Plasmid Preparation, Control, Expressing, Genomic Sequencing, Immunoprecipitation, Derivative Assay

Journal: Oncogene

Article Title: Repression of the MSP/MST-1 gene contributes to the antiapoptotic gain of function of mutant p53.

doi: 10.1038/sj.onc.1209061

Figure Lengend Snippet: Figure 3 Functional validation of shRNA expression plasmids specific for MSP and RON. (a) H1299 cells plated in 6 cm dishes and subsequently transfected with 0.5 mg of a plasmid encoding a selectable drug resistance marker (pBabePuro) together with 4.5 mg of either a control (altered pSuper p53 control) shRNA expression plasmid (cont), two different MSP-specific shRNA expression plasmids (siMSP#1 and #2, respectively) or a plasmid expressing p53R175H (Mut p53). Following 48 h of puromycin selection, cells were incubated in serum-free medium for an additional 48 h. Medium was collected and concentrated using UltraFree MC 10kd centrifuge columns (Millipore). Concentrated medium was ana- lysed by Western blot for MSP using monoclonal antibodies (R&D systems). The main specific band detected with these antibodies was the 53 kDa large cleavage product. GAPDH from the plated cells, harvested in parallel, was used as a loading control. (b) H1299 cells were transfected with a plasmid encoding puromycin resistance, along with a RON-specific shRNA expression plasmid (see Materials and methods). Drug selection was as in panel a. Cells were then harvested and total RNA was extracted. cDNA was synthesized and RON mRNA levels were analysed as described in Materials and methods.

Article Snippet: Protein sample buffer was added to the medium concentrates, which were then resolved by SDS–PAGE on a 10% polyacrylamide gel and probed with monoclonal anti-MSP antibodies (AF352, R&D systems), followed by secondary goat anti-mouse HRP-conjugated antibodies (Jackson).

Techniques: Functional Assay, Biomarker Discovery, shRNA, Expressing, Transfection, Plasmid Preparation, Marker, Control, Selection, Incubation, Western Blot, Bioprocessing, Synthesized

Journal: European journal of biochemistry

Article Title: Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator.

doi: 10.1046/j.1432-1033.2003.03549.x

Figure Lengend Snippet: Fig. 1. SDS/PAGE/Ligand blot analysis. The samples were reduced prior to electrophoresis. Left: SDS/PAGE indicates two bands of TN as a result of N-terminal cleavage. Right: Lanes 1–6 were loaded with plasminogen, tPA, uPA, HGF, prothrombin, and MSP, respectively. The blot shows TN-binding to plasminogen and HGF. However, longer exposure revealed binding to tPA as well.

Article Snippet: Ligand blot analysis using 125I-labelled tetranectin Three micrograms of bovine Plg, 4 lg human tPA, 2 lg human uPA, 2 lg human HGF (294-HGN, R&D Systems Europe, UK), 3 lg bovine prothrombin, and 2 lg human MSP (352-MS, R&D Systems) were dissolved in the Laemmli-buffer containing 2% SDS and subjected to SDS/PAGE.

Techniques: SDS Page, Electrophoresis, Binding Assay