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1.
Fig. 2.

Fig. 2. From: A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Schematic of the SKOV-3 siRNA screen and followup.

Cynthia S. Collins, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3775-3780.
2.
Fig. 4.

Fig. 4. From: A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Inhibition of MAP4K4 affects the motility of multiple carcinoma cell lines. Shown are the affects of the two most potent MAP4K4 siRNAs on the motility of SKOV-3, MDA-MB-231 (MDA-231), DU-145, ES-2, and A2058. A graphical representation of migratory inhibition relative to control siRNA is shown above RT-PCR analysis of the MAP4K4 transcript in each of the cell lines.

Cynthia S. Collins, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3775-3780.
3.
Fig. 5.

Fig. 5. From: A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Down-regulation of MAP4K4 decreases SKOV-3 cell invasion in vitro. Cells transiently transfected with siRNAs were subjected to Boyden chamber assays of cell invasion through matrigel. The data were collected from five individual consecutive fields of view (×10 objective) of each of four replicate invasion chambers. Representative photomicrographs are shown beneath the graph.

Cynthia S. Collins, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3775-3780.
4.
Fig. 6.

Fig. 6. From: A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Decrease in JNK phosphorylation after siRNA-mediated knockdown of MAP4K4. (A) Western blots of SKOV-3 total protein lysates collected 48 h after transfection with control (si_C) or MAP4K4-specific siRNAs (si_1 and si_2). Blots were probed with phospho-specific antibodies (p) and antibodies recognizing total JNK, p38, ERK, and STAT-3. (B) Scratch migration assay of SKOV-3 cells exposed to increasing concentrations of the JNK inhibitor, SP600125.

Cynthia S. Collins, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3775-3780.
5.
Fig. 3.

Fig. 3. From: A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Identification and validation of four promigratory genes. (A) Validation of migratory inhibition by phenotypic and transcriptional analysis. The migratory inhibition elicited by two independent siRNA duplexes targeting four genes, MAP4K4 (NM_004834), CDK7 (NM_001799), DYRK1B (NM_004714), and SERPINB3 (NM_006919), is shown compared to control siRNA and quantified by the automated algorithm (black bars, migration score; white bars, relative cellular viability). RT-PCR analysis is shown for each transcript, and the relative transcriptional knockdown was quantified by using imagej (National Institutes of Health). The extent of transcript knockdown is shown as a ratio to control transfected cells beneath the gel picture. (B) Four unique siRNA duplexes (three from the primary screen and one additional sequence) have similar effects on the migration of SKOV-3 cells, reducing motility by up to 75%, with a corresponding knockdown of MAP4K4 transcript ranging from 64% to 94%.

Cynthia S. Collins, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3775-3780.
6.
Fig. 1.

Fig. 1. From: A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Validation of the automated cell motility assay. (A) The temporal (0, 4, 8, 12, and 16 h) migration of SKOV-3 cells in the presence and absence of controls is shown. siCON, FITC-conjugated control siRNA; siRAC, a sequence-specific siRNA targeting RAC; DMSO, dimethyl sulfoxide; SRC, c-Src family kinase inhibitor, compound 43 (5). (B) Quantification of SKOV-3 migration. The degree of scratch closure (“migration score”) was determined by an automated algorithm and plotted as a function of time. Low migration scores reflect migratory inhibition. (C) Western blot demonstrating knockdown of the RAC protein by the RAC-specific siRNA used in A, compared to a control siRNA (CON) and mock transfected cells (LIPO). The same blot reprobed with an anti-actin antibody to demonstrate equal loading is shown below.

Cynthia S. Collins, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3775-3780.

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