Journal: The Journal of Biological Chemistry

Article Title: Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity *

doi: 10.1074/jbc.M115.689950

Figure Lengend Snippet: ExoT/GAP intoxication increases pro-apoptotic proteins at the mitochondrial membrane. HeLa cells were treated with PBS (Mock) or infected with GAP-expressing ΔU/T(G+A−) or the T3SS mutant (pscJ) at an M.O.I. of ∼10. A, 5 h after infection, the cells were fractionated, and the cytoplasmic (Cyto) and mitochondrial (Mito) fractions were probed by Western blot for the pro-apoptotic proteins Bax, Bid, and Bim and the anti-apoptotic proteins Bcl-2 and Bcl-xL. GAPDH was used as loading control for cytoplasmic fraction, and CoxIV was used as loading control for mitochondrial fraction. Note that infection with ΔU/T(G+A−) results in substantial increases in Bax and Bid and to a lesser extent Bim in the mitochondrial membrane fraction. B and C, HeLa cells were infected as in A. Five hours after infection, the cells were fixed and analyzed for Bax expression by determining the mean fluorescent intensity (MFI) and for subcellular localization of Bax (green) by IF microscopy. Representative images are shown in B, and the tabulated results from 16 random fields are shown in C. MitoTracker (red) was used as mitochondrial marker. (* indicates significance with p < 0.0001, χ2 test relative to ΔU/T(G+A−). Note that in cells infected with ΔU/T(G+A−), Bax expression is increased. Bax also forms globular complexes in mitochondria (indicated by the arrow), as indicated by its colocalization with MitoTracker. Scale bar = 10 μm.

Article Snippet: The sources of antibodies (either mouse (Ms), or rabbit (Rb)) used in these studies are as follows: cytochrome c (Cell Signaling Technology; 12959; Ms); Bax (Cell Signaling Technology; 2774; Rb) for Western blot; Bax (Abcam; ab5714; Ms) for immunofluorescence; Bid (Cell Signaling Technology; 2002; Rb); Bim (Cell Signaling Technology; 2933; Rb); Bcl-2 (Cell Signaling; 2876; Rb); Bcl- xL (Cell Signaling Technology; 2764; Rb); CoxIV (Cell Signaling Technology; 11967; Ms); JNK (Cell Signaling Technology; 9252; Rb); phospho-JNK (Cell Signaling Technology; 4668; Rb); c-Jun (Cell Signaling Technology 9165; Rb); phospho-c-Jun (Cell Signaling Technology; 3270; Rb); GAPDH (Sigma Life Sciences; G9545; Rb); caspase-9 (Cell Signaling Technology; 9508; Ms); active caspase-9 (Cell Signaling Technology; 9505; Rb); caspase-3 (Cell Signaling Technology; 9668; Ms); and active caspase-3 (Cell Signaling Technology; 9661; Rb).

Techniques: Membrane, Infection, Expressing, Mutagenesis, Western Blot, Control, Microscopy, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PAK5-mediated phosphorylation and nuclear translocation of NF-κB-p65 promotes breast cancer cell proliferation in vitro and in vivo

doi: 10.1186/s13046-017-0610-5

Figure Lengend Snippet: Impact of PAK5 on the expression of cell cycle regulatory proteins. a , b Western blot analysis of the PAK5 gene overexpression and silencing on Myc, PAK5, Cyclin D1, Cyclin D3, Cyclin A, Cyclin A1, Cyclin B1 and Cyclin B3 in BT549 and MDA-MB-231 cells. c , d Western blot analysis of the PAK5 gene overexpression and silencing on p21 and p27 in BT549 and MDA-MB-231 cells. Data are shown as mean ± SD for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Antibodies against the following proteins were used: PAK5 (1:500; Abcam); p65 (1:500; Santa Cruz); β-actin (1:1000; Zhongshan biotech); Cyclin D1, c-Myc, Cyclin B1, Cyclin B3, Cyclin A1, Cyclin A, p21, p27 (1:200; Santa Cruz); Cyclin D3 (1:2000; Cell Signaling Technology, Beverly, MA, USA); Anti-rabbit HRP, Anti-Mouse HRP (1:10,000, Vicmed).

Techniques: Expressing, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PAK5-mediated phosphorylation and nuclear translocation of NF-κB-p65 promotes breast cancer cell proliferation in vitro and in vivo

doi: 10.1186/s13046-017-0610-5

Figure Lengend Snippet: PAK5 interacts and phosphorylates p65. a MDA-MB-231 cells were transfected with pcDNA3.1-Myc-control plasmids and pcDNA3.1-Myc-PAK5 plasmids. Lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Myc and anti-p65 antibody. b MDA-MB-231 cells were transfected with pcDNA3.1-Myc-control plasmids, pcDNA3.1-p65 plasmids and pcDNA3.1-Myc-PAK5 plasmids. Lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Myc, and anti-p65 antibody. c MDA-MB-231 cells transfected with pcDNA3.1-Myc-control plasmids and pcDNA3.1-Myc-PAK5 plasmids. Then concentrated protein was used for WB. The top p-p65 represents the mobility shift of phosphorylated p65 proteins in SDS-PAGE with polyacrylamide-bound Mn 2+ -Phos-tag. The bottom p65 represents non-phosphorylated counterpart in SDS-PAGE with polyacrylamide-bound Mn 2+ -Phos-tag. d , e Luciferase reporter assay was carried out in BT549 and MDA-MB-231 cells. The relative luciferase activities of pcDNA3.1 + pGL3-basic, pcDNA3.1-p65 + pGL3-basic and pcDNA3.1-p65 + pGL3-Cyclin D1 groups normalized against pcDNA3.1 + pGL3-Cyclin D1 group. Data are shown as mean ± SD for three independent experiments. **, P < 0.01

Article Snippet: Antibodies against the following proteins were used: PAK5 (1:500; Abcam); p65 (1:500; Santa Cruz); β-actin (1:1000; Zhongshan biotech); Cyclin D1, c-Myc, Cyclin B1, Cyclin B3, Cyclin A1, Cyclin A, p21, p27 (1:200; Santa Cruz); Cyclin D3 (1:2000; Cell Signaling Technology, Beverly, MA, USA); Anti-rabbit HRP, Anti-Mouse HRP (1:10,000, Vicmed).

Techniques: Transfection, Control, Immunoprecipitation, Mobility Shift, SDS Page, Luciferase, Reporter Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PAK5-mediated phosphorylation and nuclear translocation of NF-κB-p65 promotes breast cancer cell proliferation in vitro and in vivo

doi: 10.1186/s13046-017-0610-5

Figure Lengend Snippet: PAK5 promotes the tumor development of mice. a Xenograft tumor formation of MDA-MB-231 cells in nude mice. b, c The volume and weight of tumor cancer models were analyzed and calculated. d Western blotting analyzed the protein levels of PAK5, p65 and Cyclin D1 in harvested tumor tissues. e Representative images PAK5 and p65 immunohistochemical staining with PAK5 antibody in control and PAK5 OE groups tumor models. *, P < 0.05

Article Snippet: Antibodies against the following proteins were used: PAK5 (1:500; Abcam); p65 (1:500; Santa Cruz); β-actin (1:1000; Zhongshan biotech); Cyclin D1, c-Myc, Cyclin B1, Cyclin B3, Cyclin A1, Cyclin A, p21, p27 (1:200; Santa Cruz); Cyclin D3 (1:2000; Cell Signaling Technology, Beverly, MA, USA); Anti-rabbit HRP, Anti-Mouse HRP (1:10,000, Vicmed).

Techniques: Western Blot, Immunohistochemical staining, Staining, Control