Journal: Molecular Plant Pathology

Article Title: The noncanonical type III secretion system of X anthomonas translucens pv. graminis is essential for forage grass infection

doi: 10.1111/mpp.12030

Figure Lengend Snippet: Genes encoding predicted type III secreted effectors found in the genome of X anthomonas translucens pv. graminis strain 29 ( X tg 29) and putative functions or homologues from other pathogens containing a type III secretion system (e.g. P seudomonas syringae , R alstonia solanacearum or Y ersinia spp.)

Article Snippet: , XTG29_00200 , + , Homology to HopR , Xanthomonas outer protein AM ( X. vesicatoria ATCC 35937) , 0.0 , 55 , 5166 , Wei et al . ( 2007 ).

Techniques: Sequencing

Journal: Molecular Plant Pathology

Article Title: The noncanonical type III secretion system of X anthomonas translucens pv. graminis is essential for forage grass infection

doi: 10.1111/mpp.12030

Figure Lengend Snippet: (a) Mean areas under the disease progress curve (AUDPCs) from four replications for L olium multiflorum genotype LmK‐01 infected with Xanthomonas translucens pv. graminis strain 29 (X tg29) deficient in genes encoding the type III secretion system (T3SS) compared with the wild‐type strain. Means with different letters are significantly different (P < 0.05) on the basis of a two‐sided t‐test using the Holm P value adjustment. The negative control treatment consisted of cutting the plants with scissors dipped in sterile physiological sodium chloride solution. (b) Colonization of L . multiflorum by X tg29 and the X tg29 strains deficient in T3SS genes. Bacterial population densities were determined for leaves of three or four different plants of LmK‐01 sampled at 6 h post‐infection (hpi) and 4, 7 and 14 days post‐infection (dpi), counting the colony‐forming units (CFU) of serial dilutions. For each time point, mean population densities and standard deviations were calculated. Means indicated with different letters are significantly (P < 0.05) different on the basis of a two‐sided t‐test using the Holm P value adjustment.

Article Snippet: , XTG29_00200 , + , Homology to HopR , Xanthomonas outer protein AM ( X. vesicatoria ATCC 35937) , 0.0 , 55 , 5166 , Wei et al . ( 2007 ).

Techniques: Infection, Negative Control

Journal: Oncotarget

Article Title: Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA stability

doi:

Figure Lengend Snippet: (A and B) Cl41 cells were treated with TPA/EGF (40 ng /ml) alone, or pretreated with ISO (50 μM) for 30 min and then co-incubated with ISO and TPA/EGF for indicated time periods. The cell extracts were subjected to Western blot analysis with specific antibodies against PHLPP1, PP2A-A, PP2A-B, p-PP2A, PTEN, and MKP-1. (C and D) Cl41 cells were transfected with the siMKP-1 or nonsense constructs. Stable transfectants were established and cell extracts were subjected to Western blot analysis with specific antibody against MKP-1(C). The indicated transfectants were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with ISO (50 μM) for 30 min and then co-incubated with ISO and TPA/EGF for 24 hours. The cell extracts were subjected to Western blot analysis with specific antibodies against MKP-1, cyclin D1, p-c-JunSer63, p-c-JunSer73, c-Jun, p-JNK, JNK, p-MKK7, and MKK7 (D). (E and G) Cl41 cells were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM of ISO for 30 min and then co-incubated with ISO and TPA/EGF for 48 hours as described in “Materials and Methods”. Data represent one of three different experiments as indicated in Flow cytometric analysis of cell cycle distribution (E) and percentage of cell-cycle phase (G). (F and H) The cell transformation was determined using the indicated Cl41 stable transfectants in the presence of TPA/EGF (40 ng /ml)with or without ISO (50 μM). Representative images of colonies of transformed cells in soft agar assay were presented (F), and the colonies were counted under microscopy and presented as colonies per 10,000 cells from three independent experiments (H). The symbol (*) indicates a significant increase as compared with that in nonsense transfectants treated with TPA/EGF alone ( P <0.05).

Article Snippet: The antibodies specific against cyclin D1, c-Fos, CREB, Fra1, p-NF-kB p65, NF-kB p50, JunD, JunB, PTEN and MKP-1 were bought from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Western Blot, Transfection, Construct, Transformation Assay, Soft Agar Assay, Microscopy

Journal: Oncotarget

Article Title: Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA stability

doi:

Figure Lengend Snippet: (A) Cl41 cells were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50μM of ISO for 30 min and then co-incubated with ISO and TPA/EGF for 12 hours. RT-PCR was performed to determine mkp-1 mRNA levels. The gapdh mRNA levels were used as loading control. (B) Cl41 cells stably transfected with MKP-1 promoter luciferase reporter were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM of ISO for 30 min followed by co-incubation with ISO and TPA/EGF for 24 hours. The luciferase activity was measured as described in “Materials and Methods”. The results were presented as relative MKP-1 promoter activity compared with that of the cells treated with TPA/EGF. The symbol (*) indicates a significant decrease as compared with cells treated with TPA/EGF alone ( P <0.05). (C) Cl41 cells were pretreated with ISO (50 μM) for 6 hours and then co-incubated with ISO and actinomycin D (20 μM) for indicated time periods. Total RNA was isolated and RT-PCR was then performed to determine mkp-1 mRNA levels. The result was a representative one from three independent experiments. (D) The relative mRNA level of mkp-1 was determined by ImageQuant 5.2 (GE Healthcare). Natural logarithm of ratio [ mkp-1 ] t /[ mkp-1 ] 0 was plotted against the time and the half life of mkp-1 mRNA was calculated via linear regression. (E) Cl41 cells were treated with ISO (50 μM) for indicated time periods. The total cell extracts were subjected to Western blot analysis with specific antibodies against HNRPD, VHL, Hur, Nucleolin, and MKP-1. (F) The diagram indicates mechanisms responsible for ISO inhibition of cell transformation.

Article Snippet: The antibodies specific against cyclin D1, c-Fos, CREB, Fra1, p-NF-kB p65, NF-kB p50, JunD, JunB, PTEN and MKP-1 were bought from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Luciferase, Activity Assay, Isolation, Western Blot, Inhibition, Transformation Assay

Journal: Oncotarget

Article Title: MKP-1 attenuates LPS-induced blood-testis barrier dysfunction and inflammatory response through p38 and IκBα pathways

doi: 10.18632/oncotarget.12823

Figure Lengend Snippet: TM4 Mkp-1 silencing cells and TM4 control cells were either not stimulated or stimulated with LPS (100 ng/ml). Cells were harvested for protein after 0, 15, 30, 60 and 120 minutes (short timepoint, A .) or 24 hours (long timepoint, B .). Cell lysates were subjected to western blot analysis for MKP-1, phospho-IκBα (p-IκBα), total-IκBα (IκBα), phospho-p38 (p-p38), total-p38 (p38), and β-actin (short timepoint, A) or MKP-1, occludin, and β-actin (long timepoint, B). Immunoblots were scanned, and the intensity of bands was quantified by the image program. Data were normalized to the control and expressed as the percentage of maximum activation or fold change of stimulation (a1-a3, b1-b2). C . Mkp-1 siRNA or control siRNA TM4 Sertoli cells were either not stimulated or stimulated with LPS (100 ng/ml). Cells were harvested for RNA after 1, 2 and 6 hours. Cells were harvested for RNA, and realtime PCR was carried out using primers specific to Mkp-1 (c1), occludin (c2) and β-actin . Data were normalized to the control and the ratio was expressed as fold change relative to the control sample. D . TM4 Sertoli cells were either not stimulated or stimulated with LPS. Cells were harvested for protein after 30, 60 and 120 minutes, and MKP-1 was immunoprecipitated (IP) from cell lysates and analyzed by western blotting for presence of p38, occludin and MKP-1. * p <0.05.

Article Snippet: The siRNA vectors with or without nucleotide sequences targeting Mkp-1 were obtained from Santa Cruz Biotechnology (Lafayette, CO, USA) as previously described [ ].

Techniques: Western Blot, Activation Assay, Immunoprecipitation