Journal:

Article Title: The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii -dependent modulation of JNK phosphorylation

doi: 10.1016/j.yexcr.2008.09.019

Figure Lengend Snippet: (A) A representative immunoblot of uninfected and T. gondii infected HeLa cells following exposure to UV irradiation and harvested at the time points indicated. JNK-phosphorylation (PJNK) occurs within 15 minutes post-UV and is sustained for the duration of the experiment in uninfected HeLa cells. This response is markedly inhibited both with regard to the extent of JNK phosphorylation and its kinetics in cells infected with T. gondii. Total JNK (JNK) and calnexin serve as loading controls. (B) Densitometric analysis representing the level of phospho-p54 JNK relative to total JNK in three independent experiments depicts the range of values inherent in such experiments. Relative phospho-p54 levels in uninfected cells (circle) versus infected cells (diamond) confirm a marked inhibition of mean phospho-p54 JNK accumulation (horizontal line) in parasite infected cells relative to uninfected cells. Densitometric analysis was performed as described in the materials and methods. (C) Statistical analysis using two way ANOVA indicates highly significant effects for both time post UV and infection. Time and infection as variables however do not exhibit significant interaction. Significance: p values <0.01 are considered highly significant (***), while p values between 0.01 and 0.05 are considered significant (**) and p-values > 0.05 are deemed not significant (ns).

Article Snippet: Cell and parasite maintenance HeLa (ATCC CCL-2) were maintained in Dulbecco’s Modified Eagle Medium (Gibco-Invitrogen, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum (Gemini Bio-Products, Woodland CA), 100 units ml −1 penicillin, 100 ug ml −1 streptomycin, and 2 mM L-glutamine (Gibco-Invitrogen) (DMEM).

Techniques: Western Blot, Infection, Irradiation, Phospho-proteomics, Inhibition

Journal:

Article Title: The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii -dependent modulation of JNK phosphorylation

doi: 10.1016/j.yexcr.2008.09.019

Figure Lengend Snippet: (A) A representative immunoblot of uninfected and T. gondii infected HeLa cells following exposure to TNFα and harvested at the time points indicated. JNK-phosphorylation (PJNK) occurs transiently, peaking at 30 minutes post exposure. This response is markedly inhibited in cells infected with T. gondii although the kinetics of the response do not appear to be impacted. Total JNK (JNK) and calnexin serve as loading controls. (B) Densitometric analysis representing the level of phospho-p54 JNK relative to total p54 JNK in four independent experiments depicts the range of values inherent in such experiments. Relative phospho-p54 levels in uninfected cells (open circle) versus infected cells (diamond) confirm a marked inhibition of mean phospho-p54 JNK accumulation (horizontal line) in parasite infected cells relative to uninfected cells. Densitometric analysis was performed as described in the materials and methods. (C) Statistical analysis using 2 way ANOVA indicate highly significant effects for both time post-TNFα exposure and infection. Time and infection do not exhibit significant interaction. Significance: p values of 0.01 and under are considered highly significant (***), p values between 0.01 and 0.05 are considered significant (**) while those above 0.05 are not significant (ns).

Article Snippet: Cell and parasite maintenance HeLa (ATCC CCL-2) were maintained in Dulbecco’s Modified Eagle Medium (Gibco-Invitrogen, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum (Gemini Bio-Products, Woodland CA), 100 units ml −1 penicillin, 100 ug ml −1 streptomycin, and 2 mM L-glutamine (Gibco-Invitrogen) (DMEM).

Techniques: Western Blot, Infection, Phospho-proteomics, Inhibition

Journal:

Article Title: The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii -dependent modulation of JNK phosphorylation

doi: 10.1016/j.yexcr.2008.09.019

Figure Lengend Snippet: (A) A representative immunoblot of uninfected and T. gondii-infected wild type mouse embryonic fibroblasts following exposure to UV irradiation and harvested at the time points indicated. JNK-phosphorylation (PJNK) occurs within 15 minutes post UV and is sustained for the duration of the experiment in uninfected MEF cells similar to the pattern observed in HeLa cells. This response is fundamentally identical with regard to the extent of JNK phosphorylation and its kinetics in cells infected with T. gondii. Total JNK (JNK) and calnexin serve as loading controls. (B) Densitometric of phospho-p54 JNK levels relative to total JNK in six independent experiments depicts the range of values inherent in such experiments. Relative phospho-p54 levels in uninfected cells (circle) versus infected cells (diamond) confirm an absence of inhibition of mean phospho-p54JNK accumulation (horizontal line) in parasite-infected cells relative (diamond) to uninfected cells (circle). Densitometric analysis was performed as described in the materials and methods (C) Statistical analysis using two-way ANOVA indicate a highly significant effect for time post-UV but not for infection. Time and infection do not exhibit significant interaction. The identical experiment to that performed with wild type MEF was replicated in p65−/− fibroblasts. Results indicate the absence of any significant inhibition in relative phospho-p54 JNK levels. (E) Densitometric analysis of six independent experiments confirms the broad range of responses and the lack of apparent differences in the mean levels of phospho-p54 JNK comparing uninfected (circle) and infected (diamond) p65−/− cells. (F) Two way ANOVA analysis reveals a highly significant effect for time post-UV but not infection. Significance: p values of 0.01 and lower are considered highly significant (***), p values between 0.01 and 0.05 are considered significant (**) while those of above 0.05 are not significant (ns).

Article Snippet: Cell and parasite maintenance HeLa (ATCC CCL-2) were maintained in Dulbecco’s Modified Eagle Medium (Gibco-Invitrogen, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum (Gemini Bio-Products, Woodland CA), 100 units ml −1 penicillin, 100 ug ml −1 streptomycin, and 2 mM L-glutamine (Gibco-Invitrogen) (DMEM).

Techniques: Western Blot, Infection, Irradiation, Phospho-proteomics, Inhibition

Journal:

Article Title: The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii -dependent modulation of JNK phosphorylation

doi: 10.1016/j.yexcr.2008.09.019

Figure Lengend Snippet: (A) A representative immunoblot of uninfected and T. gondii infected wild type mouse embryonic fibroblasts following exposure to TNFα and harvested at the time points indicated. JNK-phosphorylation (PJNK) occurs within 15 minutes post-UV and is transient with a pattern similar to that seen in HeLa cells. This response is fundamentally identical with regard to the extent of JNK phosphorylation and its kinetics in cells infected with T. gondii. Total JNK (JNK) and calnexin serve as loading controls. (B) Densitometric analysis representing the level of phospho-p54 JNK relative to total JNK in four independent experiments depicts the range of values inherent in such experiments. Relative phospho-p54 levels in uninfected cells (circle) versus infected cells (diamond) confirm an absence of inhibition of mean phospho-p54 JNK accumulation (horizontal line) in parasite infected cells relative (diamond) to uninfected cells (circle). Densitometric analysis was performed as described in the Materials and methods (C) Statistical analysis using two way ANOVA indicate a modest effect for time post-treatment but not for infection. Time and infection do not exhibit significant interaction.. The identical experiment to that performed with wild type MEF was replicated in p65−/− fibroblasts. Results indicate the absence of any significant inhibition in relative phospho-p54JNK levels. (E) Densitometric analysis of six independent experiments confirms the broad range of responses and the lack of apparent differences in the mean levels of phospho-p54 JNK comparing uninfected (circle) and infected (diamond) p65−/− cells. (F) Two-way ANOVA analysis reveals a highly significant effect for time post-UV but not infection. Significance: p values of 0.01 and lower are considered highly significant (***), p values between 0.01 and 0.05 are considered significant (**) while those above 0.05 are not significant (ns).

Article Snippet: Cell and parasite maintenance HeLa (ATCC CCL-2) were maintained in Dulbecco’s Modified Eagle Medium (Gibco-Invitrogen, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum (Gemini Bio-Products, Woodland CA), 100 units ml −1 penicillin, 100 ug ml −1 streptomycin, and 2 mM L-glutamine (Gibco-Invitrogen) (DMEM).

Techniques: Western Blot, Infection, Phospho-proteomics, Inhibition