Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: Increase in PAK1 expression and activation in the MCF10 progression series grown in vitro. (A) Lysates of MCF10A, NeoT, AT1, and DCIS cells were subjected to immunoblot analysis for expression of group 1 PAKs [PAK1, PAK2, and PAK3]. The membranes were stripped and reprobed for tubulin to verify equal loading. (B) Densitometric analyses of PAK1 levels from four independent experiments were normalized to the level in MCF10A cells in each experiment. Data are mean ± SEM. One-way ANOVA shows that there is a significant increase in PAK1 level (P < .05) at each progression step. (C) MCF10 series cells were cultured in the presence of 75 µM NSC23766 (Rac inhibitor) or vehicle for 8 hours, followed by a further 2 hours in serum-free medium with 1 µM okadaic acid (phosphatase inhibitor) and ± NSC23766. Lysates were Western blotted first for phosphorylated PAK1 and then the membranes were stripped and reprobed for tubulin.

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Expressing, Activation Assay, In Vitro, Western Blot, Cell Culture

Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: Expression of exogenous PAK1 constructs in MCF10 mammary epithelial progression series. (A) Cells were infected with viruses expressing exogenous myc-tagged PAK1 constructs plus EGFP, or EGFP only as the control. Cell lysates were subjected to separate Western blot assays for the myc-tag and PAK1. Overexpression of the PAK1 constructs is apparent from comparison to the signals for endogenous PAK1 in control cells exogenously expressing only EGFP. Equal loading per lane was shown by stripping and reprobing for tubulin. (B) Typical confocal fluorescent images showing equatorial slices through spheroids developed from single virus-infected cells expressing myc-tagged PAK1 constructs and coexpressing either RFP (red) or EGFP (green). Upper row: fluorescent confocal images of a spheroid cultured for 2 days; scale bar, 10 µm. Lower row: spheroid cultured for 7 days; scale bar, 50 µm. Cell nuclei were stained with DAPI (blue).

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Expressing, Construct, Infection, Western Blot, Over Expression, Stripping Membranes, Cell Culture, Staining

Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: Inhibition of the proliferation of MCF10 progression series cells by DN-PAK1. Cells were passaged onto six-well plates (0.1 x 106 cells/well) and infected with viruses expressing EGFP only (○) or EGFP plus wt-PAK1 (□), CA-PAK1 (△) or DN-PAK1 [PAK1.H83,86L, K299R (●) and PAK1.K299R (■)]. The infected cells were then passaged onto 96-well plates (1 x 103 cells/well) and cultured as shown for assay of proliferation. Data are mean ± SEM from four independent experiments.

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Inhibition, Infection, Expressing, Cell Culture

Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: Inhibition of migration/invasion of MCF10 progression series cells by DN-PAK1. Cellular migration/invasion was assayed over a 24-hour period through a gelatin/Matrigel barrier in response to 10% FBS as a chemoattractant in the lower chamber. Data are mean ± SEM from three independent experiments. One-way ANOVA shows that there is a significant (P < .05) inhibition of cell migration/invasion by DN-PAK1 in all the cell lines.

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Inhibition, Migration

Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: DN-PAK1 promotes luminal clearing in spheroids formed from cells of the MCF10 progression series in 3D rBM overlay culture. MCF10 series cells were cultured and transduced as indicated, with control retroviruses that only expressed EGFP, or bicistronic constructs expressing EGFP plus wild-type PAK1 (wt), CA-PAK1 (423E), or two forms of DN-PAK1 (83,86L,299R or 299R). The structures were fixed at the time shown, and the nuclei were stained with DAPI or DRAQ5. (A) Confocal sections at an equatorial plane through the structures are shown. Images at day 10 for MCF10A and day 15 for the other cell lines are illustrated because these were the most sensitive time points for observing the effects of PAK1 manipulation on hollow lumen formation. Data are representative of results from four independent experiments. Scale bar, 50 µm. (B) Frequencies of occurrence of hollow lumen formation (% of total structures imaged). Images of at least 50 fields were collected for each condition and analyzed for formation of hollow structures, which were defined by the absence of nuclear staining at the center of the structure. The total numbers of structures counted are listed in parentheses. To determine whether there was a significant effect of each PAK construct on the formation of hollow structures, each condition was compared to the EGFP control transduction in the same cell line by chi-square analysis (*P < .05, **P < .01).

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Cell Culture, Construct, Expressing, Staining, Transduction

Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: PAK1 regulates pericellular proteolysis in the MCF10 progression series grown in 3D rBM overlay cultures. (A and B) MCF10 series cells were infected with control retroviruses that only expressed RFP, or bicistronic constructs expressing RFP plus wild-type PAK1 (wt), CA-PAK1 (423E), or two forms of DN-PAK1 (83,86L,299R or 299R) and cultured for 2 days in Matrigel plus DQ-collagen IV. The nuclei of live cells were stained with DRAQ5 for 30 minutes, and then confocal immunofluorescent images were taken. (A) Expressions of retroviral constructs (red) and nuclei (blue) are shown in equatorial sections of the cells along with the associated cleavage of DQ-collagen IV (green). Scale bar, 10 µm. (B) Quantification of the green signals from the equatorial cross-sections of three to eight structures for each condition. The data are presented as average pixel intensity per cell, with cell number determined by counting of DRAQ5-labeled nuclei. CA-PAK1 significantly increased pericellular proteolysis, and both DN-PAK1 constructs significantly inhibited pericellular proteolysis, compared to RFP control structures, in all cell lines (P < .05). (C) Representative quantification of the green signal from 3D reconstructions of the MCF10A experiments shown in (A). The data are presented as average voxel intensity per cell from the entire structure. Cell number was determined by counting the DRAQ5-labeled nuclei in z-stacks through the structures. (D and E) DCIS cells were infected with dual sequence retroviruses that expressed RFP plus a control shRNA sequence (Ctl), or plus two forms of shRNA designed to decrease PAK1 expression (shRNA #1 and shRNA #2) and cultured for 2 days. (D) DCIS Cell lysates were subjected to immunoblot analysis for PAK1 to assess knock down and were stripped and reprobed for PAK2 to assess specificity. Quantification of results from two independent experiments demonstrated that the shRNA #2 sequence decreased PAK1 expression by 55%. (E) Transduced DCIS cells were cultured in Matrigel containing DQ-collagen IV for 3 days, and images were prepared from equatorial sections of the structures for cleavage of DQ-collagen IV (green), nuclei (blue), and expression of retroviral constructs (red). Data are representative of results from two independent experiments. Scale bar, 10 µm.

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Infection, Construct, Expressing, Cell Culture, Staining, Labeling, Sequencing, shRNA, Western Blot

Journal:

Article Title: p21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-Dimensional Culture Model for Premalignant Progression of Human Breast Cancer 1 2

doi:

Figure Lengend Snippet: A Rac/PAK1 pathway regulates pericellular proteolysis in the MCF10 progression series grown in 3D rBM overlay culture. (A) MCF10 series cells were cultured for 2 days in Matrigel/DQ-collagen IV with or without 75 µM NSC23766. Confocal fluorescent images taken at the equatorial plane of structures show live cell nuclei (blue; DRAQ5 staining) and pericellular proteolysis (green). Scale bar, 10 µm. (B) Quantification of the green signals from the equatorial cross-sections of three to nine structures for each condition. The data are presented as average pixel intensity per cell, with cell number determined by counting of DRAQ5-labeled nuclei. Inhibition of Rac greatly inhibits pericellular proteolysis in all cell lines (P < .05). (C) MCF10 series cells were infected with control retroviruses that only expressed RFP, or bicistronic constructs expressing RFP plus wild-type PAK1 (wt), CA-PAK1 (423E), or two forms of DN-PAK1 (83,86L,299R or 299R) and were cultured for 2 days in Matrigel containing DQ-collagen IV in the presence of 75 µM NSC23766. The data are equatorial sections of the cells and show cleavage of DQ-collagen IV (green), the nuclei (blue), and the expression of retroviral constructs (red). Scale bar, 10 µm. (D) Quantification of the green signals from the equatorial cross-sections of three to eight structures for each condition. The data are presented as average pixel intensity per cell, with cell number determined by counting of DRAQ5-labeled nuclei. Expression of CAPAK1 significantly increases pericellular proteolysis in all cell lines that had been treated with NSC23766 (P < .05).

Article Snippet: To knock-down PAK1 expression, PAK1 siRNA hairpin oligonucleotide sequences and a nonspecific shRNA control sequence (BD Clontech) were separately cloned into RNAi-Ready pSIREN-RetroQ-DsRed-Express retrovirus vector (BD Clontech) according to the manufacturer's instructions.

Techniques: Cell Culture, Staining, Labeling, Inhibition, Infection, Construct, Expressing