Proteomic Signatures in Thapsigargin-Treated Hepatoma Cells

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca2+-store depletion within the ER and simultaneously increases Ca2+ level in the cytosol. Perturbation of Ca2+ homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway

uPAR expression controls cell migration.

<p>uPAR-293 and V-293 cells efficiently migrate toward serum growth factors or EGF (GF). However, uPAR, when expressed, recruits and bridges fMLFRs and β1 integrin at the cell surface, thus driving pro-migratory signaling (upper panels). In fact, anti-uPAR antibodies (anti-uPAR), FPR1 desensitization by the W peptide (W Pep), inhibition of uPAR/β1 integrin interaction by P-25 peptide (P-25) or of specific cell signalling mediators block migration of uPAR-293 cells without affecting migration of uPAR-negative V-293 control cells (lower panels).</p

Proteomic Signatures in Thapsigargin-Treated Hepatoma Cells

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca2+-store depletion within the ER and simultaneously increases Ca2+ level in the cytosol. Perturbation of Ca2+ homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway

Proteomic Signatures in Thapsigargin-Treated Hepatoma Cells

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca2+-store depletion within the ER and simultaneously increases Ca2+ level in the cytosol. Perturbation of Ca2+ homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway

fMLF receptors and β1 integrins are involved in uPAR capability to control cell migration.

<p>uPAR-293 cells (<b>A and C</b>) or V-293 cells (<b>B and D</b>) were pre-incubated with diluent (-) or W Peptide (W Pep) (<b>A and B</b>), or with diluent (-) or P-25 peptide (<b>C and D</b>). Cells were then plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left panels). The values are the mean±SD of three experiments performed in triplicate. (*) p≤0.05, as determined by the Student's <i>t</i> test. Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR recruits fMLF receptors and integrins on the cell membrane.

<p><b>A:</b> HEK-293 cells were seeded on glass coverslips and transiently transfected with EGFP-uPAR cDNA; after 24 h, cells were incubated for further 24 h in DMEM containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep) in DMEM. All samples were then fixed, incubated with the anti-FPR1 polyclonal antibody and with the mouse anti-β1 monoclonal antibody, stained with Cy5 or Alexa 594 conjugated secondary antibodies, and analyzed by confocal microscopy. <b>B:</b> The degree of co-localization of the fluorescent signals in panel A was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of pixels corresponding to co-localizing uPAR-FPR1 has been normalized to green (uPAR) or blue (FPR1) pixels and reported in 1^st and 2^nd set of columns, respectively. The number of pixels corresponding to co-localizing uPAR-β1 integrin has been normalized to green (uPAR) or red (β1 Integrin) pixels and reported in 3^rd and 4^th set of columns, respectively. The number of pixels corresponding to co-localizing FPR1-β1 integrin has been normalized to blue (FPR1) or red (β1 Integrin) pixels and reported in 5^th and 6^th set of columns, respectively. (*) p≤0.05, as determined by the Student's <i>t</i> test. <b>C:</b> HEK-293 cells were seeded on glass coverslips and transiently transfected with the empty pEGFP-N1 vector; after 24 h, cells were incubated for further 24 h in DMEM containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep) in DMEM. All samples were then fixed, incubated with the anti-FPR1 polyclonal antibody and with the rabbit anti-β1 polyclonal antibody, stained with Cy5 or Alexa 594 conjugated secondary antibodies, and analyzed by confocal microscopy. <b>D:</b> The degree of co-localization of the fluorescent signals in panel C was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of pixels corresponding to co-localizing FPR1-β1 integrin has been normalized to blue (FPR1) or red (β1 integrin) pixels and reported in the graph.</p

FPR1 co-localizes with uPAR on the surface of uPAR-expressing HEK-293 cells.

<p>HEK-293 cells stably transfected with uPAR cDNA (uPAR-293 cells) were seeded on glass coverslips and transiently transfected with FPR1 cDNA. After 24 h, cells were incubated for further 24 h in culture medium (DMEM) containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep). All samples were then fixed, incubated with the anti-uPAR monoclonal R4 antibody and the anti-FPR1 polyclonal antibody (<b>A</b>) or with the anti-uPAR monoclonal R4 antibody and the rabbit anti-β1 polyclonal antibody (<b>B</b>), stained with Cy3 or Alexa 488 conjugated secondary antibodies, and analyzed by confocal microscopy. The insets show a 4× magnification for the indicated region in each merge image. <b>C:</b> The degree of co-localization of the fluorescent signals was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of yellow pixels, corresponding to co-localizing uPAR-FPR1, has been normalized to green (uPAR) or red (FPR1) pixels shown in A and reported in 1^st and 2^nd set of columns, respectively. The number of pixels corresponding to co-localizing uPAR-β1 integrin has been normalized to green (uPAR) or red (β1 Integrin) pixels shown in B and reported in 3^rd and 4^th set of columns, respectively. (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR expression controls cell migration toward EGF.

<p>Stably-transfected uPAR-293 cells (<b>A, C, E</b>) or V-293 cells (<b>B, D, F</b>) were pre-incubated with nonimmune Ig (<b>-</b>) or anti-uPAR or anti-uPAR_84–95 polyclonal antibodies (<b>A and B</b>), with diluents (-) or P-25 or W (W Pep) peptides (<b>C and D</b>), with diluents (-) or inhibitors of Rho- or Rac1-dependent signaling pathways (<b>E and F, left panels</b>), with diluents (-) or inhibitors of PI3K or ERK-MAPKs (<b>E and F, right panels</b>). Cells were then plated in Boyden chambers and allowed to migrate toward 100 ng/ml EGF. Migrated cells were fixed, stained with hematoxylin, and counted; results are expressed as percentage of cells migrated towards EGF over the cells migrated without EGF; 100% values represent cell migration in the absence of chemoattractants. The values are the mean±SD of three experiments performed in triplicate. (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR depletion or blocking impairs migration of uPAR-expressing cells.

<p><b>A:</b> Prostate carcinoma (PC3) cells were transfected with a uPAR-targeting siRNA or a non-targeting control siRNA; then, cells were partly lysed for Western blot analysis with a uPAR-specific antibody (left) and partly loaded in Boyden chamber and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (middle panel). The values are the mean±SD of a representative experiment performed in triplicate. Results of the migration assay are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractant (right). (*) p≤0.05, as determined by the Student's <i>t</i> test. <b>B:</b> PC3 cells were pre-incubated with nonimmune immunoglobulins (Ig) or anti-uPAR polyclonal antibodies, plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left). Results of the migration assay are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractant (right). The values are the mean±SD of three experiments. (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

Structural basis of antiviral activity of peptides from MPER of FIV gp36 - Fig 3

Confocal microscopy images of MLEVs in presence of NBD labelled C8 (left) and C6a (right) peptides. MLEVs vary in DOPC/DOPG composition moving from DOPC/DOPG 100:0 M/M (top) to DOPC/DOPG 0:100 M/M (bottom). Images were acquired on a laser scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging) equipped with a plan Apo 63X, NA 1.4 oil immersion objective lens. For each field, both fluorescent and transmitted light images were acquired on separate photomultipliers and were analysed with Zeiss LSM 510 4.0 SP2 software.</p