Journal: Science signaling

Article Title: The 4E-BP–eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes

doi: 10.1126/scisignal.aad8463

Figure Lengend Snippet: (A) Purified CD4+ T cells (left) and B cells (right) from wild-type (WT) C57/B6 mouse splenocytes were labeled with CFSE, pretreated with vehicle, 20 nM rapamycin (Rap), or 50 nM MLN0128 (128), and then were stimulated with anti-CD3 and anti-CD28 antibodies (for T cells) or with anti-IgM antibody and IL-4 (for B cells) for the indicated times. Cells were then fixed, permeabilized, and stained with anti-pS6 antibody to assess mTORC1 activity. Red numbers in each plot indicate the percentage of cells that stained positive for pS6. Data are representative of four independent experiments. (B) Top: The growth of the indicated cells at 24 hours after stimulation was measured by flow cytometric analysis of forward scatter (FSC). Bottom: The decrease in size compared to that of control cells was measured by calculating the percentage median fluorescent intensity (MFI) of FSC normalized to the stimulated condition for each experiment. Data are means ± SEM of three to seven experiments. (C) The proliferation of the indicated cells at 48 hours after stimulation was measured by flow cytometric analysis of CFSE dilution. Data are representative of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, by repeated-measures analysis of variance, measured versus the medium control.

Article Snippet: The mTOR allosteric inhibitor, rapamycin, was purchased either from LC Labs or Cell Signaling Technologies.

Techniques: Purification, Labeling, Staining, Activity Assay, Control

Journal: Science signaling

Article Title: The 4E-BP–eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes

doi: 10.1126/scisignal.aad8463

Figure Lengend Snippet: (A) CD4+ T cells (left) and B cells (right) from wild-type (WT) or S6K1/2−/− (DKO) mice were left unstimulated (Media) or were stimulated for 24 hours with anti-CD3 and anti-CD28 antibodies (for T cells) or with anti-IgM antibody and IL-4 (for B cells) in the presence or absence of 20 nM rapamycin (Rap). The cells were then analyzed by flow cytometry to detect pS6 (Ser240/244). Red numbers inside the plots indicate the percentage of pS6-positive cells. Data are representative of two experiments. (B) Cell growth at 24 hours was measured by flow cytometric analysis of FSC. Data are representative of two experiments (C) Cell proliferation at 72 hours after activation was measured by flow cytometric analysis of CFSE dilution. (D) Total splenocytes (left) or purified CD4+ T cells (right) from WT or S6K1−/− mice were labeled with CFSE and stimulated for 72 hours with anti-CD3 and anti-CD28 antibodies with or without the indicated concentration of PF-4708671 (PF). Cells were stained with anti-CD4 and proliferation was measured by flow cytometric analysis of CFSE dilution gating on CD4+ cells. All results are representative of two independent experiments. In addition to the comparisons of wild-type (WT) and S6K1/2−/− (DKO) mice in (A) to (C), equivalent results were observed in independent experiments using additional chemical and genetic approaches (figs. S1 and S2).

Article Snippet: The mTOR allosteric inhibitor, rapamycin, was purchased either from LC Labs or Cell Signaling Technologies.

Techniques: Flow Cytometry, Activation Assay, Purification, Labeling, Concentration Assay, Staining

Journal: Science signaling

Article Title: The 4E-BP–eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes

doi: 10.1126/scisignal.aad8463

Figure Lengend Snippet: (A) Top: B cells isolated from WT, 4E-BP1 knockout (4E-BP1−/−), and 4E-BP2 knockout (4E-BP2−/−) mice were left unstimulated or were stimulated for 2 hours with anti-IgM antibody and IL-4 in the absence or presence of 20 nM rapamycin (Rap) or 50 nM MLN0128 (128). Samples were then analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots are representative of two to six experiments. Bottom: Densitometric analysis of the intensities of the indicated bands normalized to that of the unstimulated condition. Data are means ± SEM of six independent experiments. (B) B cells from WT mice were left unstimulated or were stimulated as described earlier in the absence or presence of the indicated inhibitors. Cells were then analyzed by Western blotting with antibodies against the indicated proteins. Right: Densitometric analysis of the intensities of the band corresponding to p4EBP1 (S65) normalized to that of unstimulated condition. Data are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, by repeated-measures analysis of variance, measured versus the vehicle-treated control.

Article Snippet: The mTOR allosteric inhibitor, rapamycin, was purchased either from LC Labs or Cell Signaling Technologies.

Techniques: Isolation, Knock-Out, Western Blot, Control

Journal: Science signaling

Article Title: The 4E-BP–eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes

doi: 10.1126/scisignal.aad8463

Figure Lengend Snippet: (A) CD4+ T cells and B cells from control (R26-rtTA) mice and 4E-BP1M expressing mice were left untreated or were stimulated as described earlier for 12 hours and subjected to m7GTP cap pulldown. The relative amounts of eIF4G, eIF4E, and 4E-BP1 bound to the cap were measured by Western blotting. Data are representative of two experiments. (B) Top: The growth of the indicated 4E-BP1M–expressing lymphocytes was determined by flow cytometric analysis of FSC. Different color-coding represents the different amounts of doxycycline (dox) that were added for 6 hours before cell stimulation to induce 4E-BP1M expression. Bottom: The growth of 4E-BP1M-expressing lymphocytes was compared to that of Raptor-Δ lymphocytes. Data are representative of three experiments. (C) The proliferation of the indicated 4E-BP1M-expressing lymphocytes was compared with that of Raptor-Δ lymphocytes, and mTORC1 activity was assessed by the flow cytometric measurement of pS6 abundance as described in Fig 1A. Data are representative of three experiments. (D) Syngeneic C57/B6 host mice were fed Dox in their drinking water ad libitum for 24 hours. Purified CD4+ T cells from either control (R26-rtTA) or 4E-BP1M mice were labeled with eFluor 450 and injected intravenously into syngeneic host mice. After 24 hours, mice were injected intraperitoneally with 100 μg of SEB, and spleens were analyzed at 24 and 48 hours after injection to determine the size (left) and proliferation (right) of CD4+Vβ8+ cells. Data are means ± SEM of three mice of each genotype. *P < 0.05; **P < 0.01; ***P < 0.001, by repeated-measures analysis of variance, measured versus the media control. (E) Proposed model: Cell growth and proliferation are coupled through the 4E-BP–eIF4E pathway in primary lymphocytes, which distinguishes these cells from cells, such as fibroblasts, in which both processes are regulated in a largely independent manner by S6Ks and 4E-BP–eIF4E signaling. This coordination of cell growth and proliferation through a common 4E-BP–eIF4E node renders primary lymphocytes particularly sensitive to rapamycin, which potently blocks the phosphorylation of 4E-BP2, an isoform that is highly abundant in these cells.

Article Snippet: The mTOR allosteric inhibitor, rapamycin, was purchased either from LC Labs or Cell Signaling Technologies.

Techniques: Control, Expressing, Western Blot, Cell Stimulation, Activity Assay, Purification, Labeling, Injection, Phospho-proteomics

Journal:

Article Title: Effects of bisindolylmaleimide PKC inhibitors on p90 RSK activity in vitro and in adult ventricular myocytes

doi: 10.1038/sj.bjp.0706210

Figure Lengend Snippet: Effects of U0126 and rapamycin on the phosphorylation status of ERK1/2, p90RSK, eEF2K and eEF2 following adenovirus-mediated expression of caMEK1. ARVM were maintained in culture for 42 h, following a 1 h infection with empty virus (Cont) or adenovirus encoding caMEK1, both at an MOI of 50 PFU cell−1. ARVM were then exposed to vehicle (Veh), 1 μM U0126 (UO) or 100 nM rapamycin (RAP) for 4 h, before being lysed in SDS–PAGE sample buffer for subsequent Western immunoblot analysis. (a) Representative Western immunoblots showing the expression of MEK1 and phosphorylated forms of ERK1/2 (P-ERK1/2), p90RSK(P-p90RSK), eEF2K (P-eEF2K) and eEF2 (P-eEF2). Total ERK2 expression is also shown to illustrate equal protein loading. Quantitative data (panels b–e) illustrate the phosphorylation status of (b) ERK1/2, (c) p90RSK, (d) eEF2K and (e) eEF2, *P<0.05 versus Cont, †P<0.05 versus Veh (n=6).

Article Snippet: GF109203X, Ro31-8220, U0126 (an inhibitor of MEK1) and rapamycin (an inhibitor of the mammalian target of rapamycin (mTOR)) were from Merck Biosciences, Nottingham, U.K. and were dissolved in DMSO to prepare stock solutions.

Techniques: Expressing, Infection, SDS Page, Western Blot

Journal: The Journal of Neuroscience

Article Title: Protein Translation in the Nucleus Accumbens Is Dysregulated during Cocaine Withdrawal and Required for Expression of Incubation of Cocaine Craving

doi: 10.1523/JNEUROSCI.2412-17.2018

Figure Lengend Snippet: Incubated cocaine seeking is attenuated by blocking protein translation in the NAc. a, b, Timelines for Experiments 1–3. c, Self-administration training data for rats destined for Experiment 1 (n = 15), Experiment 2 (n = 9), or Experiment 3 (n = 16). Nose-pokes in the active hole resulted in an intravenous infusion of saline or cocaine (0.5 mg/kg) paired with a light cue. d–i, Anisomycin or rapamycin was infused into the NAc core 1 h before a cue-induced seeking test on WD1-3 or ≥WD40. During the test, nose-pokes in the active hole (termed “active responses” in y axis labels of panels d and f) delivered the cue previously associated with cocaine but no cocaine infusion. A crossover design was used so that each rat received vehicle or treatment and then treatment or vehicle 2 d (WD1 and WD3 tests) or 5 d (WD40–47 and WD45-52 tests) later. Anisomycin attenuated cue-induced cocaine seeking in late withdrawal but not early withdrawal (d) without significantly altering locomotor activity (e). Rapamycin also attenuated incubated cocaine seeking but did not alter locomotion (f). Infusion sites for the early withdrawal anisomycin experiment (Experiment 1) (g), late withdrawal anisomycin experiment (Experiment 2) (h), and rapamycin experiment (Experiment 3) (i). Data are mean ± SEM. *p < 0.05, ***p < 0.001.

Article Snippet: The mTOR inhibitor rapamycin (GenDepot) was dissolved in 100% DMSO as a stock solution and used at a final concentration of 5.47 m m in 8% DMSO/2% β-cyclodextrin in PBS.

Techniques: Incubation, Blocking Assay, Saline, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Protein Translation in the Nucleus Accumbens Is Dysregulated during Cocaine Withdrawal and Required for Expression of Incubation of Cocaine Craving

doi: 10.1523/JNEUROSCI.2412-17.2018

Figure Lengend Snippet: Examination of the mTOR pathway in NAc synaptoneurosomes prepared after prolonged withdrawal from extended-access cocaine self-administration or after a cue-induced seeking test. a, Timeline for Experiment 4. All rats underwent self-administration training (18 saline rats, 20 cocaine rats). Half were killed for NAc synaptoneurosome preparation on WD47-52 (n = 10 saline, n = 10 cocaine), and the other half underwent cue-induced seeking tests on WD47-52 and were killed 30 min later (n = 8 saline/test, n = 10 cocaine/test). b, Self-administration training. Nose-pokes in the active hole resulted in an intravenous infusion of saline or cocaine (0.5 mg/kg) paired with a light cue. c, Seeking tests on >WD47. Shown are nose-pokes in the previously active hole (active responses; a measure of cocaine seeking) during a 30 min test performed under extinction conditions (nose-poke delivers cue but no infusion). **p = 0.008 (two-tailed t test). d–h, Immunoblotting results in NAc synaptoneurosomes from saline, cocaine, saline/test, and cocaine/test groups (n = 6–9 rats/group): (d) phosphorylated (p)-Akt (Thr308) and total Akt, (e) p-mTOR (Ser2448), p-mTOR (Ser2481) and total mTOR levels, (f) p-p70s6k (Thr389) and total p70s6k, (g) p-s6 (Ser235/236) and total s6 levels, and (h) α-tubulin (loading control). For α-tubulin, we show representative images from immunoblots used to generate data for this figure and all other biochemical data, with each row indicating a lane from each of the four experimental groups (for description of how a single gel was used for multiple analyses, see Materials and Methods). Data are mean ± SEM. ****p < 0.0001.

Article Snippet: The mTOR inhibitor rapamycin (GenDepot) was dissolved in 100% DMSO as a stock solution and used at a final concentration of 5.47 m m in 8% DMSO/2% β-cyclodextrin in PBS.

Techniques: Saline, Two Tailed Test, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: Protein Translation in the Nucleus Accumbens Is Dysregulated during Cocaine Withdrawal and Required for Expression of Incubation of Cocaine Craving

doi: 10.1523/JNEUROSCI.2412-17.2018

Figure Lengend Snippet: Signaling pathways related to protein translation mechanisms. Schematic of major signaling pathways regulating protein translation, based on recent reviews (Ruggero and Sonenberg, 2005; Tai and Schuman, 2008; Costa-Mattioli et al., 2009; Liu-Yesucevitz et al., 2011; Kindler and Kreienkamp, 2012; Trinh and Klann, 2013). The directionality of phosphorylation-induced changes are as follows: eIF2α, inhibits; Akt, activates; Erk1/2, activates; 4E-BP1, activates; mTOR, activates; p70s6k, activates; s6, activates; FMRP, inhibits; eEF2, inhibits; eIF4E, activates.

Article Snippet: The mTOR inhibitor rapamycin (GenDepot) was dissolved in 100% DMSO as a stock solution and used at a final concentration of 5.47 m m in 8% DMSO/2% β-cyclodextrin in PBS.

Techniques: Protein-Protein interactions, Phospho-proteomics