Journal: PLoS Pathogens

Article Title: Rapamycin inhibits pathogen transmission in mosquitoes by promoting immune activation

doi: 10.1371/journal.ppat.1009353

Figure Lengend Snippet: (A) Influence of P . berghei infection on the TOR pathway by Western blot analysis. Phosphorylation levels of S6K from fat bodies 12 h and 24 h post-normal (NB) and infectious blood meal (IB) were analyzed using anti-Phospho-S6K antibody. An . stephensi S6K and ACTIN were used as internal controls (left panel). The intensity of phosphorylated S6K was normalized to that of S6K. The relative intensity of phosphorylated S6K in IB-fed mosquitoes was normalized to that of NB-fed mosquitoes (right panel). Results were pooled from three independent experiments. (B) Schematic overview of rapamycin microinjection of An . stephensi . Vehicle solution was injected as a control. (C) The influence of rapamycin treatment on the mosquito TOR pathway. Phosphorylation levels of S6K from fat bodies 24 h post-blood meal were analyzed. Quantification of p-S6K signal intensity in rapamycin-treated mosquitoes was normalized to that of controls. The results are from two independent experiments. (D) Influence of rapamycin treatment on Plasmodium infection. Data were pooled from three independent replicates. (E) Influence of TOR knockdown on the activity of the TOR signaling pathway. Relative quantification of p-S6K signal intensity was from two independent replicates. (F) Influence of TOR knockdown on Plasmodium infection. The data were pooled from three independent biological experiments. Horizontal black bars indicate median oocyst numbers. Each dot represents an individual mosquito. Error bars indicate standard errors. Significance was determined by Student’s t- test in (A), (C), and (E) and by Mann-Whitney tests in (D) and (F); *P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: Antibodies used for TOR signaling were rabbit anti-phospho-S6K (Thr398) (1:1000) (Cell Signaling), rabbit anti-S6K (1:1000), and rabbit anti-actin (1:2000) (Sungenebiotech, China).

Techniques: Infection, Western Blot, Injection, Activity Assay, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Rapamycin inhibits pathogen transmission in mosquitoes by promoting immune activation

doi: 10.1371/journal.ppat.1009353

Figure Lengend Snippet: (A) Schematic overview of exposing mosquitoes to a rapamycin-treated surface. A solvent-coated surface was used as a control. t E , exposure time. (B) Oocyst numbers in mosquitoes exposed to rapamycin (0.77 mmol/m 2 ) (red dots) or solvent (black dots) coated surfaces for 60 min. (C) Mosquitoes were exposed to a 0.77 mmol/m 2 rapamycin-coated Petri dish for 30 min, 10 min, and 6 min. Data were pooled from two independent replicates (B, C). (D - H) Mosquitoes were incubated with a 0.77 mmol/m 2 rapamycin- or solvent-coated surface for 10 min. (D) Survival curve of mosquitoes exposed to rapamycin (n = 38) or solvent (n = 36) coated surfaces. (E) Western blot analysis of S6K phosphorylation in fat bodies collected from rapamycin-exposed mosquitoes and controls. The bar chart represents relative quantification of signal intensity of p-S6K from two independent replicates as determined by ImageJ software. Error bars indicate standard errors. (F) Sporozoite numbers of rapamycin-treated mosquitoes (red dots) and controls (black dots). Data were pooled from two independent replicates. (G) Ovaries were dissected at 48 h post-normal blood meal from rapamycin-exposed and control mosquitoes. Scale bar, 200 μm. (H) Mean number of eggs laid by 25–35 gravid rapamycin-exposed and control mosquitoes. Data were pooled from three independent replicates. Results from one of three independent experiments are shown (D, G). Horizontal black bars indicate the median values. Significance was determined by Mann-Whitney tests in (B), (C), and (F), by a Log-rank (Mantel-Cox) test in (D), and by Student’s t -test in (E) and (H); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Antibodies used for TOR signaling were rabbit anti-phospho-S6K (Thr398) (1:1000) (Cell Signaling), rabbit anti-S6K (1:1000), and rabbit anti-actin (1:2000) (Sungenebiotech, China).

Techniques: Incubation, Western Blot, Software, MANN-WHITNEY

Journal: Cells

Article Title: Cell Type-Specific Metabolic Response to Amino Acid Starvation Dictates the Role of Sestrin2 in Regulation of mTORC1

doi: 10.3390/cells11233863

Figure Lengend Snippet: Sestrin2dependent regulation of mTORC1 upon amino acid deprivation. ( A ) Sestrin2 protein levels and phosphorylation of P70S6K after two days of amino acid deprivations; ( B ) phosphorylation of P70S6K upon SESN2 silencing in the presence or absence of indicated amino acids; ( C , D ) densitometric quantification of Western blots from three independent experiments in MCF7 cells (n = 3). ANOVA with a post hoc Tukey test was used to measure statistical significance; ( E ) Sestrin2, ATF4 levels, and the phosphorylation of P70S6K in MCF7 cells upon the cotreatment with GCN2 inhibitor (1 µM) and PERK inhibitor (1 µM) in either the presence or absence of serine (0.4 mM), glycine (0.4 mM), or in combination; ( F ) timedependent regulation of Sestrin2 and phosphorylation of P70S6K upon the serine deprivation; ( G ) protein synthesis in MCF7 cells measured with puromycin (90 µM) with CHX (20 µM) as a positive control upon siNC or siSESN2. Samples were re-probed for the vinculin control. Dots represent individual values. ** and *** indicate p ≤ 0.01 and 0.001, respectively. n.s. indicates statistical insignificance.

Article Snippet: Primary antibodies used in this study: Sestrin2 (Proteintech, Planegg-Martinsfried, Germany), total AMPKα, total p38, Actin, Vinculin (Santa Cruz Inc, Heidelberg Germany), phospho-P70S6K (R&D Systems, McKinley Place NE Minneapolis, USA), phospho-AMPKα, total P70S6K, ATF4, and phospho-p38 (Cell Signaling Technology Europe, Frankfurt am Main, Germany).

Techniques: Western Blot, Positive Control

Journal: Scientific Reports

Article Title: Xuefu Zhuyu decoction, a traditional Chinese medicine, provides neuroprotection in a rat model of traumatic brain injury via an anti-inflammatory pathway

doi: 10.1038/srep20040

Figure Lengend Snippet: ( A , B ) The levels of p-AKT decreased significantly on the 1 st and 3 rd days in ipsilateral brain tissue after TBI. Treatment with 9 g/kg XFZY significantly reduced the increase in p-AKT expression from the 1 st to the 14 th day compared with the Vehicle group. The same trend was observed in the 18 g/kg XFZY group on the 1 st , 3 rd and 14 th days. The expression levels of p-AKT were significantly lower in the 9 g/kg XFZY group compared with the 18 g/kg XFZY group on the 7 th and 14 th days. No significant changes were detected in total AKT expression between the Sham and Vehicle group. ( C , D ) p-mTOR was significantly increased from the 1 st to the 14 th day after TBI. Treatment with 9 g/kg XFZY significantly decreased p-mTOR expression compared with the Vehicle group from the 1 st to the 14 th day. Treatment with 18 g/kg XFZY significantly reduced p-mTOR on the 1 st and 3 rd days. mTOR expression in the 9 g/kg XFZY group was significantly reduced compared with the 18 g/kg XFZY group on the 1 st , 7 th and 14 th days. There were no significant changes in total mTOR in comparisons between each group. ( E , F ) p-p70S6K increased significantly from the 1 st to the 14 th day after TBI. Treatment with 9 g/kg XFZY significantly reduced p-p70S6K expression on the above days. The same tendency was observed in the 18 g/kg XFZY group on the 1 st day. p-p70S6K expression was significantly reduced in the 9 g/kg XFZY group compared with the 18 g/kg XFZY group on the 3 rd , 7 th and 14 th days. No significant changes were detected in total p70S6K expression between the Sham and Vehicle group. Data are the mean ratio between targeting proteins and β-actin and are presented as the percentage of XFZY (or Vehicle)-treated brains over the control Sham-treated brains. The data represent the mean fold induction ± SEM, as analyzed by ANOVA. *p < 0.05, # p < 0.01 vs. the Vehicle group. ∆ p < 0.05, □ p < 0.01 vs. the 9 g/kg XFZY group.

Article Snippet: The membranes were incubated with the following primary antibodies: AKT (1:2,000; Cell Signaling Technology, Boston), phospho-AKT (1:2,000; Cell Signaling Technology, Boston), mTOR (1:1,000; Cell Signaling Technology, Boston), phospho-mTOR (1:100; Cell Signaling Technology, Boston), phospho-p70S6K (1:200; Santa Cruz Biotechnology, California), p70S6K (1:1,000; Cell Signaling Technology, Boston) and β-actin (1:4,000; Santa Cruz Biotechnology, California).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Xuefu Zhuyu decoction, a traditional Chinese medicine, provides neuroprotection in a rat model of traumatic brain injury via an anti-inflammatory pathway

doi: 10.1038/srep20040

Figure Lengend Snippet: Following TBI, the mechanical injury-stimulated cell membrane releases arachidonic acid (AA), which is metabolized into prostaglandin E2 (PGE2) and prostacyclin (PGI2) by cyclooxygenase-2 (COX-2) and into leukotrienes (LTB 4 ) by 5-lipoxygenase (5-LO). The three inflammatory mediators initiate acute inflammation, including changes in blood flow, increased capillary permeability and inflammatory cell recruitment in the brain injury ambitus zone, including polymorphonuclear leukocytes (i.e., neutrophils) and monocytes. Excess prostaglandins and leukotrienes contribute to chronic inflammation. The neutrophils are activated to further release chemotactic factors, devour necrotic tissue and sterilize bacteria. The influx of monocytes and resident microglial cells develop into macrophages, which secrete pro-inflammatory factors (e.g., TNF-α and IL-1β) and consume foreign bodies, necrotic tissue or apoptotic cells (e.g., efferocytosis). XFZY significantly suppressed the increased levels of blood AA, TNF-α and IL-1β in brain tissue, indicating that XFZY possesses anti-inflammatory effects. To target the PI3K-AKT-mTOR signaling pathway, XFZY significantly reversed the elevated phosphorylation of AKT/mTOR in brain tissue post-TBI, as well as the downstream p70S6K, resulting in a reduced translation ratio of inflammatory factors and exerting anti-inflammatory effects.

Article Snippet: The membranes were incubated with the following primary antibodies: AKT (1:2,000; Cell Signaling Technology, Boston), phospho-AKT (1:2,000; Cell Signaling Technology, Boston), mTOR (1:1,000; Cell Signaling Technology, Boston), phospho-mTOR (1:100; Cell Signaling Technology, Boston), phospho-p70S6K (1:200; Santa Cruz Biotechnology, California), p70S6K (1:1,000; Cell Signaling Technology, Boston) and β-actin (1:4,000; Santa Cruz Biotechnology, California).

Techniques: Permeability

Journal: The Journal of Neuroscience

Article Title: Translational Control via the Mammalian Target of Rapamycin Pathway Is Critical for the Formation and Stability of Long-Term Fear Memory in Amygdala Neurons

doi: 10.1523/JNEUROSCI.4209-06.2006

Figure Lengend Snippet: Training-related increase in phospho-p70s6K is specific to the learned association. Bars represent mean optical density (OD) values (±SEM) expressed as a percentage of control. a, b, There were no changes in total-p70s6K expression after immediate or delayed shock (a), but a significant increase in phospho-p70s6K was seen only in rats that received a delayed shock (b). Immunofluorescence images (20× magnification) from single subjects killed from their home cage (HC) (c) or given a single shock immediately (IMM) or 2 min after placement into the chamber (DLY) are shown. d, e, Although controls (d) received the same shock and stimulus exposure, phosphorylation of the enzyme in the lateral amygdala was seen only in rats that formed new long-term memory for fear conditioning (e) (*p < 0.05).

Article Snippet: Slices were incubated overnight in 500 μl of primary antibody diluted 1:50 against phospho-p70s6K (Thr 412) (Upstate Biotechnology).

Techniques: Expressing, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Translational Control via the Mammalian Target of Rapamycin Pathway Is Critical for the Formation and Stability of Long-Term Fear Memory in Amygdala Neurons

doi: 10.1523/JNEUROSCI.4209-06.2006

Figure Lengend Snippet: Time-dependent activation of phospho-p70s6K in the amygdala after fear conditioning. Bars represent mean optical density (OD) values (±SEM) expressed as a percentage of control. a, Rats killed at 30 and 60 min after fear conditioning showed significantly more phospho-p70s6K expression in the amygdala compared with home cage (HC) controls or animals killed 10 min after conditioning. This increase in phospho-p70s6K seen 60 min after training is reduced by an immediate posttraining injection of RAP (b). c, d, No differences between groups were observed when the same samples were probed with an antibody for total p70s6K (c), and no changes in total p70s6K expression were seen in the amygdala after RAP infusion (d) (*p < 0.05).

Article Snippet: Slices were incubated overnight in 500 μl of primary antibody diluted 1:50 against phospho-p70s6K (Thr 412) (Upstate Biotechnology).

Techniques: Activation Assay, Expressing, Injection