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The extracellular‐signal‐regulated‐kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT‐007a (GT‐7), an anti‐cancer compound, stimulates ERK... more
The extracellular‐signal‐regulated‐kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT‐007a (GT‐7), an anti‐cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT‐7 stimulates ERK phosphorylation and induces apoptosis in ERK‐active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT‐7 in T3M4 cells. The immunoblotting showed that GT‐7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase‐3 was observed only after 2‐h incubation with GT‐7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho‐ERK) in the nucleus upon 1‐h incubation with GT‐7. Fractionation experiments showed that GT‐7 increases ...
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Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in... more
Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in fission yeast. atg1 deletion cells (Δ atg1 ) exhibit the vic (viable in the presence of immunosuppressant and Cl - ) phenotype, indicative of Pmk1 signaling inhibition. Moreover, the Δ atg1 Δ pmk1 double mutant resembles the single Δ pmk1 mutant, suggesting that Atg1 functions in the Pmk1 pathway. In addition, the growth defect induced by overexpression of Pck2, an upstream activator of Pmk1 MAPK was alleviated by the deletion of atg1 + . Finally, the deletion of autophagy-related genes recapitulates Pmk1 MAPK signaling inhibition. Our data suggest a novel role for autophagy in MAPK signaling regulation.
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The mitogen-activated protein kinase (MAPK) pathway is a highly conserved eukaryotic signalling cascade that converts extracellular signals into various outputs, such as cell growth and differentiation. MAPK is phosphorylated and... more
The mitogen-activated protein kinase (MAPK) pathway is a highly conserved eukaryotic signalling cascade that converts extracellular signals into various outputs, such as cell growth and differentiation. MAPK is phosphorylated and activated by a specific MAPK kinase (MAPKK): MAPKK is therefore considered to be an activating regulator of MAPK. Pmk1 is a MAPK that regulates cell integrity and which, with calcineurin phosphatase, antagonizes chloride homeostasis in fission yeast. We have now identified Pek1, a MAPKK for Pmk1 MAPK. We show here that Pek1, in its unphosphorylated form, acts as a potent negative regulator of Pmk1 MAPK signalling. Mkh1, an upstream MAPKK kinase (MAPKKK), converts Pek1 from being an inhibitor to an activator. Our results indicate that Pek1 has a dual stimulatory and inhibitory function which depends on its phosphorylation state. This switch-like mechanism could contribute to the all-or-none physiological response mediated by the MAPK signalling pathway.
Research Interests: Biology, Enzyme Inhibitors, Cell Biology, Medicine, Multidisciplinary, and 12 moreMAP Kinase pathway and its regulation, Nature, Signal Transduction, Protein Kinases, Mitogen Activated Protein Kinase, Kinase, Schizosaccharomyces Pombe, Amino Acid Sequence, Cell Cycle Proteins, Phosphorylases, Molecular Sequence Data, and Mitogen- Activated Protein Kinases
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Fission yeast its3(+) encodes an essential phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) that regulates cell integrity and cytokinesis. We performed a genetic screen to identify genes that function in PI4P5K-mediated signaling, and... more
Fission yeast its3(+) encodes an essential phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) that regulates cell integrity and cytokinesis. We performed a genetic screen to identify genes that function in PI4P5K-mediated signaling, and identified gyp10(+) encoding a Rab GTPase-activating protein (GAP), a negative regulator for Rab GTPase signaling. Its3 overproduction caused growth defects and abnormal cytoplasmic accumulation of the Its3 protein, which can be stained by calcofluor. Notably, Its3 overproducing cells displayed abnormal membranous structures, multilamella Golgi and fragmented vacuoles showed by Electron microscopy. Furthermore, the excess cytoplasmic Its3 structure partly colocalized with the fluorescence of FM4-64. Gyp10 rescued both growth defects and abnormal Its3 localization when it was over-expressed. Gyp10 functionally interacted with the Rab GTPases Ypt3 and Ryh1, both of which regulate Golgi membrane trafficking. Consistently, mutation or deletion of Ypt3 and Ryh1 suppressed phenotypes associated with Its3 overproduction. Importantly, the plasma membrane localization of Its3 was also affected by the impairment of the Ypt3/Ryh1 Rab membrane trafficking, thus suggesting that membrane trafficking events regulated by two Rab GTPases functionally interacts with PI4,5P2 signaling. These results suggest a mechanism whereby PI4P5K signaling/localization is affected by Golgi membrane trafficking, thus provide a functional link between the PI4,5P2 signaling and Rab-mediated trafficking.
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Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase... more
Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the p...
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The expression of oncogene products and growth factors (epidermal growth factor, transforming growth factor-beta, erbB-2, ras p 21, and c-myc) in gallbladder cancer and chronic cholecystitis was measured by immunohistochemical staining on... more
The expression of oncogene products and growth factors (epidermal growth factor, transforming growth factor-beta, erbB-2, ras p 21, and c-myc) in gallbladder cancer and chronic cholecystitis was measured by immunohistochemical staining on paraffin-embedded serial sections. Expression of these products was graded according to staining intensity in an area of positively stained cells. This study reports the detection of oncogene products and growth factors in cholecystitis as well as in early and late gallbladder cancer. The multiexpression of oncogene products and growth factors was greater for both gallbladder cancer groups as compared with the cholecystitis group. The percentage of epidermal growth factor positivity diminished with increased proportion of interstitial tissue and, conversely, the percentage of transforming growth factor positivity increased with increased proportion of interstitial tissue. The proportion of ras positivity was significantly greater in both early and advanced cholecystic cancer as compared with cholecystitis, but also was considerable even for cholecystitis. These results suggest that various oncogenes may have significant roles in gallbladder cancer and that collagen synthesis is reduced by epidermal growth factor and enhanced by transforming growth factor-beta.
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The regulation of cytoplasmic Ca is crucial for various cellular processes. Here, we examined the cytoplasmic Ca levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using... more
The regulation of cytoplasmic Ca is crucial for various cellular processes. Here, we examined the cytoplasmic Ca levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca 2+ caused a dose-dependent increase in the cytoplasmic Ca level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burstlike peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress t...
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<p>(A) Analysis of Nrd1-GFP localization under stress. Localization of Nrd1-GFP in living cells grown at 27°C (untreated) after a shift to 42°C for 20 min (42°C 20 min) and after exposure to 2.0 mM arsenite (2.0 mM arsenite 120... more
<p>(A) Analysis of Nrd1-GFP localization under stress. Localization of Nrd1-GFP in living cells grown at 27°C (untreated) after a shift to 42°C for 20 min (42°C 20 min) and after exposure to 2.0 mM arsenite (2.0 mM arsenite 120 min), 5.0 mM H<sub>2</sub>O<sub>2</sub> (5.0 mM H<sub>2</sub>O<sub>2</sub> 30 min), 10.0 mM CdCl<sub>2</sub> (10.0 mM CdCl<sub>2</sub> 120 min), or 1.0 M KCl (1.0 M KCl 10 min) for the times indicated. Wild-type (wt) cells transformed with pREP1-GFP-Nrd1 were grown in EMM (thiamine-free medium) for 18 h to induce overproduction of GFP-Nrd1 (overproduction 18 hr). Bar, 10 µm. The number in the picture indicates the SG number/cell in each experiment. Right panel: Quantitative analysis of the number of SGs/cell on each stress. Graph depicting the number of stress granules per cell formed before (untreated) and after each condition as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g001" target="_blank">Figure 1(A)</a> plotted against time after exposure to each stress and the <i>inset</i> is a magnification of the results obtained on KCl treatment. (B) Co-localization of Nrd1 with poly(A)-binding protein (Pabp). Merged image of fluorescence micrographs showing Pabp-GFP (green) and Nrd1-tdTomato (red) in untreated cells and after a 20-min incubation at 42°C. Bar, 10 µm. (C) Fluorescence micrographs of the wild-type cells expressing mCherry-tagged Nrd1 grown at 26°C (untreated); and these were subjected to <i>in situ</i> hybridization with a digoxigenin-labeled oligo (dT)<sub>50</sub> probe after a 40-min exposure to 2.0 mM arsenite (2.0 mM arsenite 40 min). The hybridized probe was detected by treatment with mouse anti-digoxin antibody, followed by a fluorescein-conjugated goat anti-mouse IgG antibody (FITC). Nrd1 was detected using mCherry fluorescence (mCherry-Nrd1). Nuclei are counterstained using DAPI dye (DAPI). Bar, 10 µm. (D) Cycloheximide (CHX) prevents the formation of heat-shock- and arsenite-induced Nrd1 granules. Fluorescent images of cells expressing Nrd1-GFP incubated (from left to right) at 27°C with 100 µg/ml CHX for 30 min (CHX); at 42°C for 20 min; pre-incubated with CHX for 30 min at 27°C followed by 20-min incubation at 42°C (CHX then 42°C 20 min); 20-min incubation at 42°C followed by CHX incubation (42°C 20 min then CHX); with 2.0 mM arsenite for 120 min at 27°C; pre-incubated with CHX for 30 min followed by 120-min incubation with 2.0 mM arsenite; and 120-min pre-incubation with arsenite followed by 30-min incubation at with CHX. Bar, 10 µm. Lower panel: Graph depicting the number of stress granules per cell in each condition plotted against time after exposure to each stress.</p
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<p>(A) Schematic representation of Sip1 protein, <i>Sip1-i4</i> mutant protein and Sip1ΔN protein. The Sip1 protein is 1919 amino acids long and contains HEAT repeats (black). Star represents termination codon at the... more
<p>(A) Schematic representation of Sip1 protein, <i>Sip1-i4</i> mutant protein and Sip1ΔN protein. The Sip1 protein is 1919 amino acids long and contains HEAT repeats (black). Star represents termination codon at the amino acid position 1434 found in the <i>sip1-i4</i> allele. The <i>Sip1-i4</i> mutant protein lacks the C-terminal 485 amino acids. The Sip1ΔN protein lacks the N-terminal 1414 amino acids. (B) Binding assay involving <i>Sip1-i4</i> and the 4 subunits of the AP-1 complex. GST pull-down experiments were performed using <i>Sip1-i4</i>-GST, <i>Sip1-i4</i>-GST expressed under the control of the <i>nmt1</i> promoter. Cells that expressed GFP alone or GFP-tagged to the 4 subunits of the AP-1 complex were harvested, and their lysates were incubated with the purified <i>Sip1-i4</i> fused GST protein. GST-tagged proteins were analyzed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068488#pone-0068488-g003" target="_blank">Figure 3B</a>. (C) Binding assay involving Sip1ΔN and the 4 subunits of the AP-1 complex. The binding assay was performed as described in B. (D) Binding assay involving <i>Sip1-i4</i> and various mutant forms of Rho3. GST pull-down experiments were performed using <i>Sip1-i4</i>-GST expressed under the control of the <i>nmt1</i> promoter. Cells that expressed GFP alone or various GFP-tagged mutant forms of Rho3 were harvested, and their lysates were incubated with the purified <i>Sip1-i4-</i>GST protein. GST-tagged <i>Sip1-i4</i> was precipitated with glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies. (E) Binding assay involving Sip1ΔN and various mutant forms of Rho3. The binding assay was performed as described in (D). Lower panel: Quantitation of GFP-tagged various mutant forms of Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels as shown in D and E. Data from at least three independent experiments are expressed as means ± standard deviations.</p
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<p>(A) Fission yeast cells are sensitive to FTY720. A serial dilution assay of the wild-type strain grown in YPD medium or YPD medium containing the indicated concentrations of FTY720 in the absence (Left: YPD) or presence (Right: +... more
<p>(A) Fission yeast cells are sensitive to FTY720. A serial dilution assay of the wild-type strain grown in YPD medium or YPD medium containing the indicated concentrations of FTY720 in the absence (Left: YPD) or presence (Right: + 100 mM CaCl<sub>2</sub>) of 100 mM CaCl<sub>2</sub>. Cells were incubated for 3 days at 27°C. (B) Quantitative measurements of cell growth in the presence of FTY720. The cells were grown in liquid YES cultures to an OD<sub>660</sub> of 0.3 and were treated with the drugs (FTY720) at the concentrations indicated, and the quantitative measurements of cell growth rates were performed using a microplate reader (Sunrise<sup>TM</sup> series, Tecan, Switzerland). A representative for three independent curves is presented. (C) Addition of CaCl<sub>2</sub> exacerbated the fission yeast sensitivity to FTY720. Wild-type cells were cultured in YES liquid medium and treated with 100 mM CaCl<sub>2</sub> in the absence or presence of indicated concentrations of FTY720, and the growth curve of the cells were shown by measuring OD<sub>660</sub> for 10 h. (D) Graph shows the OD<sub>660</sub> at 10 h of the cells, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g001" target="_blank">Figure 1(B) and (C)</a>. The data were averaged from three independent experiments. Bars, SD.</p
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<p>(A) Disassembly of stress-induced Nrd1 granules. Wild-type cells expressing GFP-tagged Nrd1 were grown in EMM+thiamine at 27°C. After a 20-min incubation at 42°C, the cells were allowed to recover for 60-min by incubating them at... more
<p>(A) Disassembly of stress-induced Nrd1 granules. Wild-type cells expressing GFP-tagged Nrd1 were grown in EMM+thiamine at 27°C. After a 20-min incubation at 42°C, the cells were allowed to recover for 60-min by incubating them at 27°C (recovery from 42°C 60 min). After a 15-min exposure to 2.0 mM arsenite at 27°C (2.0 mM arsenite 15 min), the cells were washed and allowed to recover for 30- (2.0 mM arsenite 45 min) or 240-min (recovery from arsenite 240 min). After a 30-min exposure to 5.0 mM H<sub>2</sub>O<sub>2</sub> at 27°C, the cells were washed and allowed to recover for 30 min (recovery from H<sub>2</sub>O<sub>2</sub> 30 min). After a 120-min exposure to 10.0 mM CdCl<sub>2</sub> at 27°C, the cells were washed and allowed to recover for 60 min (recovery from CdCl<sub>2</sub> 60 min). After a 10-min exposure to 1.0 M KCl at 27°C, the cells were washed and allowed to recover for 30 min (recovery from KCl 30 min). After a 60-min exposure to 1.0 M KCl, Nrd1-positive granules resolved (1.0 M KCl 60 min). Bar, 10 µm. Lower panel: Graphs showing the number of stress granules per cell from each strain after each condition as indicated. (B) Δ<i>nrd1</i> cells displayed transient stress sensitivity. Wild-type cells transformed with control vector or the <i>nrd1</i><sup>+</sup> genes, or the Δ<i>nrd1</i> cells transformed with control vector were grown to mid-log-phase in EMM+thiamine at 27°C. The indicated cells were then exposed to thermal stress (48°C 90 min), 2.0 mM arsenite for 120 min, 5.0 mM H<sub>2</sub>O<sub>2</sub> for 30 min, 10.0 mM CdCl<sub>2</sub> for 180 min, and 1.0 M KCl for 180 min and were then spotted onto YES plates and incubated at 27°C. (C) CPU assay of the cells as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g006" target="_blank">Figure 6(B)</a>. Cells were treated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g006" target="_blank">Figure 6(B)</a>, and the colony forming ability of each strain after each condition as indicated was determined by counting the number of viable colonies and normalized to the number of colonies in unstressed condition for each strain. This experiment is representative of two independently performed experiments.</p
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<p>(A) Nrd1 binds to Cpc2 in a phosphorylation-dependent manner. Cells expressing endogenous Cpc2-GFP were transformed with plasmids harboring GST alone, GST-Nrd1, GST-Nrd1<sup>DD</sup>, or... more
<p>(A) Nrd1 binds to Cpc2 in a phosphorylation-dependent manner. Cells expressing endogenous Cpc2-GFP were transformed with plasmids harboring GST alone, GST-Nrd1, GST-Nrd1<sup>DD</sup>, or GST-Nrd1<sup>AA</sup> and were grown to mid-log-phase in normal medium (EMM) at 27°C (untreated) and were exposed to a 10-min incubation at 42°C (heat shock), or 120-min incubation to 2.0 mM arsenite at 27°C (arsenite). Cell lysates (lysate) and proteins bound to glutathione sepharose (pull-down) were analyzed by immunoblotting using anti-GFP antibodies (lysates, and pull-down), and anti-GST antibodies (pull-down). (B) Effects of Cpc2 deletion on granule formation of Nrd1 and Nrd1<sup>DD</sup> after heat shock or arsenite stress. GFP-Nrd1 or GFP-Nrd1<sup>DD</sup> localization in the wild-type cells (wt) or Δ<i>cpc2</i> cells under the conditions indicated. Bar, 10 µm. Lower panel: Graphs showing the number of stress granules per cell from each strain. (C) Overproduction of Nrd1 induced dot-like structures in the absence of stress in a phosphorylation- and Cpc2-dependent manner. Wild-type (wt) or Δ<i>cpc2</i> cells transformed with pREP1-GFP-Nrd1 or pREP1-GFP-Nrd1<sup>DD</sup> were grown in EMM (thiamine-free medium) for 16 h, 18 h, or 22 h to induce overproduction of GFP-Nrd1 or GFP-Nrd1<sup>DD</sup>. Bar, 10 µm. (D) Immunoblot of GFP-Nrd1 from each strain after each condition as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g004" target="_blank">Figure 4(C)</a>. Lower panel: Quantification of Nrd1 protein levels against tubulin at each condition as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g004" target="_blank">Figure 4(D)</a>.</p
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<p>(A) Left: Monitoring of intracellular Ca<sup>2+</sup> levels in wild-type (wt) cells harboring <i>adh1</i>-GFP-19-AEQ (pKB6892) treated with various concentrations of FTY720 at a time point of 5 min and... more
<p>(A) Left: Monitoring of intracellular Ca<sup>2+</sup> levels in wild-type (wt) cells harboring <i>adh1</i>-GFP-19-AEQ (pKB6892) treated with various concentrations of FTY720 at a time point of 5 min and the luminescence was followed for 1 h. An aequorin assay was performed as described in the Materials and Methods. The data shown are the representative of multiple experiments. Right: Graph shows the average of peak heights from three independent experiments shown in the left column of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>. Bars, SD. (B) Effects of EGTA and CaCl<sub>2</sub> on the FTY720-induced increase in the cytoplasmic Ca<sup>2+</sup> level. The experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>, except that prior to the addition of FTY720, 20 mM EGTA (left) or 100 mM CaCl<sub>2</sub> (middle) were added to the EMM medium. Right: The histogram was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4A</a>. (C) (D) Effects of EGTA and CaCl<sub>2</sub> on the FTY720-induced increase in the calcineurin activity. The experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3 (B)</a> with wt 3×CDRE, except that prior to the addition of 10 μM FTY720, 10 mM or 20 mM EGTA or 200 mM CaCl<sub>2</sub> were added to the EMM medium. The histogram was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3 (B)</a>.</p
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<p>(A) The colocalization of GFP-Rho3 with FM4-64 in wild-type cells. Wild-type (wt) cells, expressing chromosome-bone GFP-Rho3 under the control of the <i>nmt1</i> promoter, were examined by fluorescence microscopy... more
<p>(A) The colocalization of GFP-Rho3 with FM4-64 in wild-type cells. Wild-type (wt) cells, expressing chromosome-bone GFP-Rho3 under the control of the <i>nmt1</i> promoter, were examined by fluorescence microscopy under the repressed conditions. The cells were incubated with FM4-64 fluorescent dye for 5 min at 27°C to visualize Golgi/endosomes. The fluorescence of the adaptin subunit and FM4-64 was examined under the fluorescence microscope. Arrows indicate the plasma membrane and medial region. Arrowheads indicate the dot-like structures and Golgi/endosomes. Bar 10 µm. (B) The partial colocalization of GFP-Rho3 with Apm1-mCherry in wild-type cells. Wild-type cells expressing chromosome-borne GFP-Rho3 were transformed with pREP1-Apm1-mCherry. The cells were cultured and observed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#pone-0016842-g006" target="_blank">Figure 6</a> (A). Bar: 10 µm.</p
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<p>(A) Rho3 suppressed the defective localization of GFP-Syb1 in Δ<i>apm1</i> cells. Wild-type cells (wt) and Apm1-deletion (Δ<i>apm1</i>) expressing chromosome-bone GFP-Syb1 cells transformed with pDB248 or... more
<p>(A) Rho3 suppressed the defective localization of GFP-Syb1 in Δ<i>apm1</i> cells. Wild-type cells (wt) and Apm1-deletion (Δ<i>apm1</i>) expressing chromosome-bone GFP-Syb1 cells transformed with pDB248 or the vector containing <i>rho3<sup>+</sup></i> was cultured in YPD medium at 27°C. The GFP-Syb1 localization was examined under the fluorescence microscope. Bar 10 µm. (B) Rho3 suppressed the defective localization of FM4-64 in Δ<i>apm1</i> cells. Wild-type (wt) and Apm1-deletion cells (Δ<i>apm1</i>) transformed with pDB248 or the vector containing <i>rho3<sup>+</sup></i> were cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye for 5 min, resuspended in water, and examined by fluorescence microscopy. Bar 10 µm. (C) Wild-type (wt) and Apm1-deletion cells (Δ<i>apm1</i>) transformed with pDB248 or the vector containing <i>rho3<sup>+</sup></i> cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye for 60 min, resuspended in water, and examined by fluorescence microscopy. Bar 10 µm. (D) Wild-type cells and Δ<i>apm1</i> cells, which were transformed with either the pDB248 vector or the <i>rho3<sup>+</sup></i>-containing vector, were assayed for acid phosphatase activity as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#s2" target="_blank">Materials and Methods</a> section. Values from 3 independent experiments were plotted as means ± S.D.</p
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The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its... more
The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its lack of efficient therapies. We have previously identified ACAGT-007a (GT-7), an anti-cancer compound that kills ERK-active melanoma cells by inducing ERK-dependent apoptosis. Here, we investigated the apoptosis-inducing effect of GT-7 on three PDAC cell lines and its relevance with the MAPK/ERK and PI3K/AKT signaling pathways. GT-7 induced apoptosis in PDAC cells with different KRAS mutations (MIA-Pa-Ca-2 (KRAS G12C), T3M4 (KRAS Q61H), and PANC-1 (KRAS G12D)), being T3M4 most susceptible, followed by MIA-Pa-Ca-2, and PANC-1 was most resistant to apoptosis induction by GT-7. GT-7 stimulated ERK phosphorylation in the three PDAC cells, but only T3M4 displayed ERK-activation-dependent apoptosis. Furthermore, GT-7 induced a marked down-regulation of A...