Blood.
Extra large 
|
|
|
Figure 5. I{{kappa}}B-{{alpha}} is stabilized by As in an IKK-independent manner. Effects of As (1 µM), IFN (1000 IU/mL), combined As/IFN, or MG132 (5 µM) on phospho-I{{kappa}}B-{{alpha}} (A) and I{{kappa}}B-{{alpha}} (B) protein levels in HuT-102 cells at different time points. For I{{kappa}}B-{{alpha}} expression, the upper band (large arrow) represents the 36-kDa major I{{kappa}}B-{{alpha}} protein, whereas the lower bands of 34 and 32 kDa (small arrows) presumably represent N-terminal proteolytic forms of I{{kappa}}B-{{alpha}} up-regulated by As/IFN treatment.
- Accordingly, the use of an antibody recognizing the phosphorylated form of IB- indicated that phosphorylation of IB- was not affected by As, IFN, or As/IFN, except at very late time points (48 hours with As/IFN; Figure 5A).
- Nevertheless, despite its ongoing phosphorylation by IKKs, IB- is clearly stabilized by As or As/IFN (Figure 5B).
- Two protein species with a lower molecular weight20 were always observed after 48 hours of As/IFN treatment (small arrows in Figure 5B).
- The delayed decrease in phospho-IB- levels on As/IFN treatment (Figure 5 and Mahieux et al21) was probably due to this proteolysis.
- Importantly, the magnitude of stabilization by As was much greater for IB- than for IB- (Figures 5B and 6), possibly suggesting that IB- plays a dominant role in As-induced RelA cytoplasmic sequestration and consequently in shutting off the NF-B pathway.
Blood.
Extra large 
|
|
|
Figure 6. I{{kappa}}B-{{epsilon}} degradation by the proteasome is blocked by As exposure in HTLV-1+ cells. Effects of As, IFN, As/IFN combination, or MG132 on I{{kappa}}B-{{epsilon}} protein levels in HuT-102 cells after 48 hours of exposure to these drugs. Equal protein loading was assessed by hybridization with antiactin or anti-GAPDH antibodies.
- MG-132 stabilized IB- dramatically in the HTLV-1–transformed cells only (Figure 6 and not shown), suggesting that, as in the case of IB-, Tax-activated, IKK-dependent, IB- phosphorylation triggers IB- proteasomal degradation.
- Strikingly, like IB-, IB- protein was stabilized markedly by As in HTLV-1–infected HuT-102 and MT-2 cells but not in HTLV-1– cells (Figure 6 and not shown).
- Note that MG-132 and As/IFN similarly stabilized IB- and did not have additive effects (Figure 6B), suggesting that As inhibits the proteasomal degradation of IB-.
- Importantly, the magnitude of stabilization by As was much greater for IB- than for IB- (Figures 5B and 6), possibly suggesting that IB- plays a dominant role in As-induced RelA cytoplasmic sequestration and consequently in shutting off the NF-B pathway.
The Journal of biological chemistry.
Extra large 
|
|
|
Fig. 7. Model for regulation of bile acid biosynthesis by FGFR4. A, the biosynthesis of primary bile acids from cholesterol occurs by two pathways. The classical neutral pathway for bile acid synthesis is rate-limited by Cyp7a, and the acidic pathway is initiated and rate-limited by Cyp27a. Oxysterols, which are proportional to animal cholesterol levels, activate LXR{alpha}, which activates transcription of Cyp7a. Bile acids activate FXR, which represses Cyp7a transcription. FGFR4 and FXR repress Cyp7a expression by different mechanisms (see "Discussion"). A magnified depression of Cyp27a expression in knockout mice due to dietary cholate may indicate a positive role of FGFR4 (Fig. 6A). B, multilevel repression of Cyp7a expression by FGFR4 and FXR through activation of co-repressor RIP140. Depression of RIP140 expression in mice on high cholesterol may indicate that LXR{alpha} also down-regulates RIP140 expression (Fig. 6B).
- A knockout of Cyp7a, the rate-limiting enzyme of the classical bile acid pathway (Fig. 7A), caused reduction in bile acid synthesis, fat-soluble vitamin deficiency, and liver failure in 3 weeks in 90% of newborn mice.
- This confirmed the compensatory capability of the late onset secondary or alternate pathway of bile acid synthesis (Fig. 7A).
- These developments have revealed the exquisite metabolite-controlled transcriptional networks that balance the concentration of harmful, but essential, cholesterol and its metabolites in a physiological context (Fig. 7).
Genome Biology
Extra large 
|
|
|
Topologies of signaling networks leading to cytokine releases derived from PCR minimal models and ANOVA analysis. In each panel, nodes in the upper row represent ligands that significantly regulate respective cytokines (ANOVA). Nodes in the middle row represent significant pathways identified by PCR minimal models. Edges between top and middle rows represent significant signaling pathway regulation by the given ligands (ANOVA). Edges between top and bottom rows, or middle and bottom rows, represent significant participation identified by PCR minimal models. Weak activation of signaling pathways is indicated by dashed edges. Light gray: pathways demonstrated in the literature to not play any role (false positives).
- Connections were then drawn from the ligands that significantly stimulate cytokines to the signaling pathway identified in the PCR minimal models according to activations identified by ANOVA (Figure 7).
Molecular & cellular proteomics : MCP.
Extra large 
|
|
|
FIG. 7. TNF-{{alpha}} and TNF-{{alpha}} + insulin treatments elicit quantitatively distinct signaling patterns in HT-29 cells. Endogenous ERK (green), Akt (red), JNK1 (blue), IKK (purple), and MK2 (orange) activities in HT-29 cells in response to: A, 50 ng/ml TNF-{{alpha}} (filled circles) and B, 50 ng/ml TNF-{{alpha}} + 100 nM insulin (open circles). Lysates were generated at 0, 5, 15, 30, 60, 90 min and 2, 4, 8, 12, 16, 20, 24 h, then measured for kinase activity with the high-throughput multiplex kinase assay. Results are plotted as the mean fold activation of three independent cell extracts. Error bars are omitted for clarity, but are shown in panel C. C, comparison of ERK, Akt, JNK1, IKK and MK2 activities in response to TNF-{{alpha}} (filled circles) and TNF-{{alpha}} + insulin (open circles) from A and B, plotted as mean fold activation ± S.E. of triplicate samples.
- Because much of the signaling induced by these cues occurs shortly after cytokine addition, time points were more densely sampled in the first 4 h (Fig. 7, A and B, inset).
- From these cell extracts, quantitative measurements of ERK, Akt, JNK1, MK2, and IKK kinase activity were performed using the high-throughput multiplex kinase activity assays (Fig. 7, A and B).
- As shown in Fig. 7C, the dynamics of activation of some pathways, such as the ERK pathway (Fig. 7C, green), were essentially superimposable.
- In contrast to the subtle influences of insulin on ERK, JNK1, IKK, and MK2 signaling, it was readily apparent that insulin dramatically augmented Akt activity, inducing a rapid increase within 5 min and sustaining activation for 24 h, whereas with TNF- alone, the Akt response was much smaller in magnitude (Fig. 7C, red).
- A, hypothetical two-phase contribution to the sustained Akt activity that was observed in the presence of insulin (Fig. 7, B and C).
- Thus, i) there may exist additional response-determining pathways that our measurements did not capture, or ii) the subtle, time-dependent quantitative differences we measured in the JNK1, IKK, and MK2 pathways (Fig. 7C) may modulate phenotypic response.
Molecular & cellular proteomics : MCP.
Extra large 
|
|
|
- Coating conditions for each anti-kinase antibody on Protein A/G microtiter plates were individually optimized, revealing that the antibodies maximally bound their intended targets when 50 µl of 10 µg/ml antibody was applied to each well (Fig. 2, A–D, left panels), consistent with our estimates of the number of antibody binding sites on the plate surface.
- Higher coating concentrations of polyclonal antibodies (anti-JNK1, anti-IKK/ß, and anti-MK2) reduced the solid-phase avidity for the kinase and caused a net decrease in purification efficiency (Fig. 2, B–D, left panels).
- At the optimized coating concentration (Fig. 2, A–D, left panels, arrows), the immunopurification was always linear in the amount of kinase purified over a substantial range of lysate concentrations (Fig. 3, A–D).
- To investigate the sensitivity, dynamic range, and linearity of these assays, HT-29 and HeLa cells were treated with known activators of each kinase, and the kinase activities (CPM on the PC filter) measured as a function of different dilutions of the activated lysates (Fig. 2, A–D, middle panels).
- The signal-to-noise and reproducibility characteristics for each kinase assay were examined by selecting a single concentration in the middle of the dynamic range of each assay (Fig. 2, A–D, middle panels, arrows) and comparing the kinase activities between lysates from stimulated and unstimulated cells.
- Therefore, the kinetics of the in vitro reaction were examined in detail at a selecting fixed lysate concentration within the linear range (Fig. 2, A–D, middle panels, arrows).
- Assays were incubated with fixed amounts of HT-29 or HeLa lysates treated with known activators of the pathway of interest (for details, see Fig. 2, A–D).
- At a fixed lysate concentration (Fig. 2, A–D, middle panels, arrows), a dose-dependent decrease in the measured activity (Fig. 2, A–D, right panels) was observed, consistent with the reported IC50 values for the target kinase.
Molecular & cellular proteomics : MCP.
Extra large 
|
|
|
FIG. 7. Proteins and complexes implicated in cell adhesion and cell-cell contact (A) and their modulation (B) by transient exposure to EGF (10 ng/ml 10 min) or to erlotinib (OSI-774; 1 µM for 120 min) and were modeled using present data, Network Explorer (Ingenuity), and literature data.
- Consistent with this interpretation, caveolin-1 recovery within the anti-pY fraction was reduced by EGF receptor blockade (Fig. 7B).
- Second, Src phosphorylation at Y419, required for full kinase activity, was observed by immunoblot (Fig. 7B) and was strongly suggested by LC-MS/MS (data not shown).
- However, no change in phospho-Src family Y419 (active state; Fig. 7B), phospho-Src Y530 (repressed state), or anti-pY Yes could be detected by immunoblot even after 120 min of EGF receptor kinase inhibition (data not shown).
- LPA stimulated phospho-paxillin levels while erlotinib modestly decreased paxillin phosphorylation (Figs.
- Protein complexes were modeled using the pathway analysis Network Explorer software and through literature data (Fig. 7A).
- Data from ICAT and immunoblot experiments were superimposed upon this framework to visualize points of regulation (Fig. 7B).
- Interestingly, EGF receptor kinase inhibition for 120 min elicited a similar more modest pattern of decreased recovery of focal adhesion regulating proteins within the phosphotyrosine fraction (Table IV and Fig. 7).
- Similarly, immunoblot experiments indicate p130CAS was reduced in the anti-pY fraction but not in the anti-EGF receptor fraction, consistent with a reduction in tyrosine phosphorylation state but not in EGF receptor interaction (Fig. 7B), which was underestimated by ICAT labeling (Table IV).
- Similarly, anti-pY capture of paxillin, p130CAS, and the FAK homolog Pyk2 were all reduced by EGF receptor inhibition (Fig. 7B), while no changes in the phosphorylation of ACK1, -catenin, or Tyk2 (Figs.
- 6 and 7B) were observed.
- When the EGF receptor kinase was transiently inhibited by erlotinib, a similar pattern of focal adhesion phosphoprotein decrease was observed, for example for FAK as measured by ICAT labeling and for Pyk and paxillin by immunoblot (Fig. 7B).
- This also was observed for p130CAS, where isolation within the phosphotyrosine affinity fraction was decreased by EGF receptor blockade (Fig. 7B), while interaction with EGF receptor was unchanged.
Genome Biology
Extra large 
|
|
|
Respiratory chain inhibitors induce higher levels of cell death in AD cells. (a) Light photomicrographs of SS, AD and AS cells after 72 h. (b) Distribution of mitochondria in SS (left), AD (center) and AS (right) cells. (c) Respiratory chain inhibitors rotenone (10 μM), CCCP (50 μM), antimycin A (10 μg/ml) and oligomycin (5 μg/ml) induce higher levels of cell death in AD cells, as assessed by a cell counting assay after a 24 h incubation with the specified drugs. Data are representative of three independent experiments.
- We observed that androgen starvation caused LNCaP cells to adopt a distinct neuronal morphology (Figure 4a, middle panel) when compared to steady-state (SS) or AS cells (Figure 4a, left and right panel, respectively), a finding also reported by other investigators [44].
- The cellular distribution of mitochondria in AD cells was quite distinct as the mitochondria traversed the length of the long dendritic-like processes (Figure 4b, middle panel), whereas SS and AS cells displayed a more perinuclear mitochondrial localization pattern (Figure 4b, left and right panels, respectively).
- To test whether AD cells had an increased sensitivity to pharmacological agents that specifically disrupt different steps of the Oxphos pathway when compared with AS cells, we incubated SS, AD and AS cells with rotenone (10 μM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 μM), antimycin A (10 μg/ml) or oligomycin (5 μg/ml) and determined cell viability 24 hours later (Figure 4c).
- As shown in Figure 4c, a 24 h exposure to rotenone, which specifically blocks the NADH dehydrogenase of complex I [45] elicited a slightly higher level of cell death in AD (38.1 ± 3.0%) cells when compared to AS (31.8 ± 2.0%) and SS cells (27.2 ± 1.9%).
- An additional data file (Additional data file 1) containing all ICAT protein quantifications and statistical analyses pertaining to Figures 1,2,3,4 and Table 1 presented in this study is available.
Journal of cell science.
Extra large 
|
|
|
Fig. 1. Three stereopairs of 0.20 µm thick sections of wild-type S. cerevisiae cells grown at 24°C. (a) Shows a nucleus (N) delimited by a lightly stained nuclear envelope in continuity with ER sheets or ribbons seen in side view (ER). One of these ER elements is continuous (arrow) with a strongly stained tubular network, probably a Golgi element (G). Another sheet of ER seen in face view (er) is in close proximity to another strongly stained tubular network showing more intensely stained nodular dilations (G). Intensely stained free secretion granules (SG) are interspersed throughout the cytoplasm. CW, cell wall. Magnification x18,800. (b) Lightly stained ER ribbons are seen in oblique or side views (ER). Strongly stained structures resembling secretion granules (white arrow) are observed at the left-hand extremity of an obliquely sectioned ER ribbon. Spherical membranous structures (V) resembling vacuoles are in close contact with the nuclear envelope. N, nucleus. Magnification x40,500. (c) A non-perforated ER sheet (ER) seen in face view is continuous at its periphery with lightly stained anastomosed tubules (arrows) forming a wide-meshed tubular network. At the right-hand side extremity of this network, there are more intensely stained dilations (arrowheads) that resemble secretion granules (SG). Magnification x74,000.
- In wild-type cells grown at 24°C or 37°C, the nucleus was delimited by a well delineated nuclear envelope perforated by nuclear pores (Fig. 1a,b).
- Any of these ER elements could display continuities with membranous tubules forming networks of various sizes and staining densities (Fig. 1a; Fig. 2a).
- Some of these networks showed approximately the same staining intensity as the more or less perforated sheets to which they were connected (Fig. 1c; Fig. 2a).
- Large strongly stained dilations were similar in size and staining intensity to secretion granules seen throughout the cytoplasm (Fig. 1a,b; Fig. 2d,e) and clustering within the growing bud (Fig. 2f).
- Strings of strongly stained granules were observed in proximity to the tubular networks (Fig. 1c).