Journal: FEBS letters

Article Title: Over expression of PPP2R2C inhibits human glioma cells growth through the suppression of mTOR pathway.

doi: 10.1016/j.febslet.2013.09.029

Figure Lengend Snippet: Fig. 3. PPP2R2C inhibits mTOR pathway through de phosphorylation of S6K but not 4EBP1. (A) Overexpression of PPP2R2C in glioma cells decreases phosphorylation of S6K, but phosphorylation of AKT and 4EBP1 have no significant change. (B) Cell proliferation rates at 24 h and 48 h after cells were transfected with wild-type S6K and Kinase active S6K in B55 Gamma overexpression glioma cells compared with control cells. (C) Cell viability of the B55 Gamma overexpression glioma cells and control cells after the treatments of Rapamycin. (D) Exogenous B55 Gamma does not interact with S6K but enhance the binding of PP2A-C with S6K.

Article Snippet: Wild-type S6K and kinase active S6K were purchased from Addgene (#26610, #8988: pRK7-HA-S6K1-F5A-E389).

Techniques: De-Phosphorylation Assay, Over Expression, Phospho-proteomics, Transfection, Control, Binding Assay

Journal: FEBS letters

Article Title: Over expression of PPP2R2C inhibits human glioma cells growth through the suppression of mTOR pathway.

doi: 10.1016/j.febslet.2013.09.029

Figure Lengend Snippet: Fig. 4. Knocking down PPP2R2C in NHA cells. (A) Knocking down PPP2R2C in NHA cells recovers phosphorylation of S6K and mTOR activity. (B) Knocking down PPP2R2C in NHA cells promotes cell proliferation.

Article Snippet: Wild-type S6K and kinase active S6K were purchased from Addgene (#26610, #8988: pRK7-HA-S6K1-F5A-E389).

Techniques: Phospho-proteomics, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

doi: 10.1074/jbc.m114.568659

Figure Lengend Snippet: FIGURE 1. BDNF activation of mTOR-p70S6K increases cell survival in a rapamycin-sensitive manner. A, immunofluorescent stain of neurons, red, MAP2; blue,To-Pro3.Insets,yellowarrowsindicatelivingcells;arrowheadsindicatedeadcells,andscalebarindicates50m.B,quantificationofsurvivalratesmeasured by immunofluorescence, two independent experiments, five replicates each. C, representative Western blots from cells treated for 1 h. D, quantification of p-mTOR levels normalized to control from Western blots in C, and data are from five separate experiments analyzed by repeated measures ANOVA. PD is 100 M PD98059; Wort is 0.5 M wortmannin, and Rap is 200 nM rapamycin. For all graphs, * is compared with control; # is compared with BDNF; ns, p 0.05; *, p 0.05; ** or ##, p 0.01; *** or ###, p 0.001.

Article Snippet: Plasmids containing p70S6K (32) in competent DH5a E. coli were as follows: constitutively active p70S6K (Addgene plasmid 8993 pRK7-HA-S6K1-E389- CT), dominant negative p70S6K (Addgene plasmid 8986 pRK7-HAS6K1-F5A), wild-type p70S6K (Addgene plasmid 8984 pRK7HA-S6K1-WT), and the empty plasmid (Addgene 10883 pRK7) was used as a negative control.

Techniques: Activation Assay, Staining, Immunofluorescence, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

doi: 10.1074/jbc.m114.568659

Figure Lengend Snippet: FIGURE 5. BDNF increases LC3 puncta number through both mTOR-dependent and -independent pathways. A, immunofluorescence images of neurons after 24-h survival treatment with and without Baf A1. Green, MAP2; red, LC3; blue, DAPI. The black and white images were obtained by automated LC3 puncta identification and analysis. rap, rapamycin. B, quantification of LC3 puncta number after 24-h treatment. C, quantification of individual LC3 puncta size. D, LC3 puncta flux calculated by comparison of total puncta load with or without Baf A1. Baf A1 (100 nM) was used for the last 3 h of treatment. Data are from six slides, with 10 neurons analyzed per group per slide. E, neurons transfected with p70S6K mutants were stained for LC3 and analyzed by Volocity; red, LC3; green, HA; blue, DAPI. F, quantification of LC3 puncta number from four experiments for a total of 70 WT neurons, 38 CT neurons, and 30 F5A neurons in withdrawal medium, and 67 WT neurons, 42 CT neurons, and 33 F5A neurons in BDNF-containing medium. For all graphs,* is compared with control; # is compared with BDNF; *, or #, p 0.05; **, p 0.01; scale bars, 20 m.

Article Snippet: Plasmids containing p70S6K (32) in competent DH5a E. coli were as follows: constitutively active p70S6K (Addgene plasmid 8993 pRK7-HA-S6K1-E389- CT), dominant negative p70S6K (Addgene plasmid 8986 pRK7-HAS6K1-F5A), wild-type p70S6K (Addgene plasmid 8984 pRK7HA-S6K1-WT), and the empty plasmid (Addgene 10883 pRK7) was used as a negative control.

Techniques: Immunofluorescence, Comparison, Transfection, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

doi: 10.1074/jbc.m114.568659

Figure Lengend Snippet: FIGURE 8. Hypothesized pathways for BDNF regulation of autophagy in primary neurons. In trophic factor withdrawal medium (left panel), there is low autophagic initiation and therefore low autophagic flux, but mitochondrial stress leads to apoptosis through caspase activation. If rapamycin is added to withdrawal medium, the mTOR inhibition of the final step of autophagy is released, but cells lack sufficient (mTOR-independent) autophagic initiation to provide substrates to maintain high levels of autophagic flux. In BDNF treatment (middle panel), caspase activation is suppressed and neurons experience a balance of mTOR-independent autophagic initiation and mTOR-dependent suppression of autophagosome degradation. Under conditions of mTOR suppres- sion due to rapamycin or IL-1 (right panel), mTOR-independent autophagic initiation is not restrained by p70S6K feedback and rapamycin (rap) promotes autolysosome maturation, resulting in excessive autophagic flux and cell death.

Article Snippet: Plasmids containing p70S6K (32) in competent DH5a E. coli were as follows: constitutively active p70S6K (Addgene plasmid 8993 pRK7-HA-S6K1-E389- CT), dominant negative p70S6K (Addgene plasmid 8986 pRK7-HAS6K1-F5A), wild-type p70S6K (Addgene plasmid 8984 pRK7HA-S6K1-WT), and the empty plasmid (Addgene 10883 pRK7) was used as a negative control.

Techniques: Activation Assay, Inhibition