Journal: Infection and Immunity

Article Title: The Type III System-Secreted Effector EspZ Localizes to Host Mitochondria and Interacts with the Translocase of Inner Mitochondrial Membrane 17b ^▿

doi: 10.1128/IAI.05761-11

Figure Lengend Snippet: Infection with EPEC ΔespZ causes severe loss of Δψm. (A) HeLa cells were infected with either EPEC WT, ΔespZ, or ΔespZ/espZ or left uninfected (UI). JC-1 reagent was added to cells 15 min prior to termination of infection, and cells were washed with assay buffer three times. Fluorescence intensity was measured at excitation/emission wavelengths of 560/595 nm and 485/535 nm for J monomers (low Δψm) and J aggregates (high Δψm), respectively. Δψm was plotted as the ratio of J aggregates/J monomers. Asterisks indicate statistical significance (P < 0.05) by Student's t test for samples tested in triplicate. p.i., postinfection. (B) Supernatants of HeLa cells infected for 2, 3, or 4 h with either EPEC WT, ΔespZ, or ΔespZ/espZ, left uninfected, or treated with Triton X-100 (100% cytotoxicity; data not shown) were assayed for the presence of LDH. Values were normalized against those for Triton X-100-treated cells (100% cytotoxicity) and uninfected cells (1% cytotoxicity). Asterisks indicate statistical significance (P < 0.05) by Student's t test for samples tested in triplicate. These data are representative of three independent experiments.

Article Snippet: TIM17b cDNA was obtained from clones from the BD Matchmaker pretransformed HeLa cell cDNA library as previously described ( 28 ).

Techniques: Infection, Fluorescence

Journal: Infection and Immunity

Article Title: The Type III System-Secreted Effector EspZ Localizes to Host Mitochondria and Interacts with the Translocase of Inner Mitochondrial Membrane 17b ^▿

doi: 10.1128/IAI.05761-11

Figure Lengend Snippet: EspZ localizes to host cell mitochondria. (A) HeLa cells were plated on glass coverslips and transfected with pcDNA3::HA2espZ. Cells were stained with rabbit anti-COX IV (red) and mouse anti-HA.11 (green), followed by Alexa 633-conjugated goat antirabbit and Alexa 488-conjugated goat antimouse antibodies, respectively. Coverslips were mounted in ProLong Gold with DAPI to image cellular nuclei (blue). (B) HeLa cells were transfected with either pcDNA3::HA2espZ or pcDNA3::HA2, and mitochondrial fractions were isolated as described in the text. Antibodies that recognize cytochrome c and β-tubulin were used as controls for mitochondrial (Mito) and cytoplasmic (Cyto) fractions, respectively. Anti-HA antibodies were used to visualize HA2EspZ.

Article Snippet: TIM17b cDNA was obtained from clones from the BD Matchmaker pretransformed HeLa cell cDNA library as previously described ( 28 ).

Techniques: Transfection, Staining, Isolation

Journal: Infection and Immunity

Article Title: The Type III System-Secreted Effector EspZ Localizes to Host Mitochondria and Interacts with the Translocase of Inner Mitochondrial Membrane 17b ^▿

doi: 10.1128/IAI.05761-11

Figure Lengend Snippet: siRNA knockdown of TIM17b dampens the ability of EspZ to protect HeLa cells from EPEC-mediated cell lysis. HeLa cells treated with either TIM17b-specific or nontargeting (control) siRNA were infected with EPEC WT (A) or ΔespZ (B) for 2, 3, or 4 h. LDH present in cellular supernatants was quantified and plotted as percent cytotoxicity. Asterisks indicate statistical significance of samples tested in triplicate (P < 0.05). Data are representative of two independent experiments. (C) Lysates were generated from EPEC-infected and uninfected cells and subjected to Western blot analysis using anti-TIM17b and anticalnexin (loading control) antibodies to demonstrate siRNA knockdown efficiency.

Article Snippet: TIM17b cDNA was obtained from clones from the BD Matchmaker pretransformed HeLa cell cDNA library as previously described ( 28 ).

Techniques: Knockdown, Lysis, Control, Infection, Generated, Western Blot

Journal: Oncotarget

Article Title: Glutaminase inhibition in multiple myeloma induces apoptosis via MYC degradation

doi: 10.18632/oncotarget.20691

Figure Lengend Snippet: (a) Western Blot analysis of MYC and selected candidates of the glutamine metabolism in untreated myeloma cell lines. All MYC-expressing cell lines (INA-6, RPMI-8226, OPM-2, MM.1S) show marked protein expression of glutaminase (GAC) and the substrate transporters (ASCT2, LAT1). U266 cells lack MYC protein expression along with the weakest levels of glutaminase isoform GAC and low of ASCT2 expression. (b) Growth-inhibitory effect targeting glutaminase. Enzyme inhibition was performed in a 2-fold serial dilution and measured after 5 days of incubation by MTT-assay using the small molecules 968 (n=3), its inactive control compound 335 (n=2) and BPTES (n=3). INA-6 cells are most susceptible to 968-treatment, whereas U266 cells show the weakest response to 10 μM 968 in relation to DMSO-control. BPTES reduced cell proliferation only moderately with a maximum of 40% independent of the treated cell line. (c) Comparison of INA-6 and U266 cell proliferation during 10 μM 968 treatment for 4 days analyzed by MTT. U266 proliferation was reduced by 50 % compared to DMSO-control on day four, while 968-treated INA-6 cells show no proliferation at all. (d) Treatment of INA-6 and U266 cells with 5μM 968 for 3 days reduced the percentage of viable Annexin V-FITC/PI negative cells in INA-6 by 50% whereas U266 cells showed no signs of apoptosis (n=3). Western Blot analysis on day 1 and 2 after 968 treatment confirms induction of apoptosis in INA-6 cells by PARP-1 cleavage (cl. PARP-1 indicates cleaved PARP-1 fragment). (e) Primary MM and peripheral blood mononuclear cells (PBMCs) were treated with 10μM 968 or DMSO as control for 72h and the percentage of viable cells (Annexin V-FITC negative, PI negative) compared to the DMSO treated sample were quantified using Annexin V-FITC and PI staining.

Article Snippet: Quantitative transcriptome analysis using random-primed cDNA library from endogenous cell lines INA-6, U266 and the generated MYC-positive U266-CDS-MYC cells (GATC Biotech).

Techniques: Western Blot, Expressing, Enzyme Inhibition Assay, Serial Dilution, Incubation, MTT Assay, Control, Comparison, Staining

Journal: Oncotarget

Article Title: Glutaminase inhibition in multiple myeloma induces apoptosis via MYC degradation

doi: 10.18632/oncotarget.20691

Figure Lengend Snippet: (a) Western Blot shows protein expression in U266 cells before and after lentiviral MYC-transduction (U266/U266-MYC) compared to endogenous expression levels in INA-6. MYC expression significantly elevates GAC and ASCT2 protein expression in U266 cells. (b) Oligomycin and 2-deoxyglucose (2-DG) treatment of INA-6, U266 and U266-MYC. Comparison of ATP-generation from glycolysis (2-DG incubation) and oxidative phosphorylation (oligomycin treatment) revealed that INA-6 cells synthesize ATP via glycolysis independent of oxidative phosphorylation as 2-DG reduces measurable ATP. In contrast, U266 produce ATP via mitochondrial respiration, which can be blocked using the ATP-synthase inhibitor oligomycin. INA-6 and U266 cells use contrary mechanisms to support ATP generation. U266-MYC cells are more sensitive to glycolysis inhibition and less sensitive to inhibition of the oxidative phosphorylation pathway (n=3). (c) Western Blot of MM cell lines incubated with or without glucose and L-glutamine in the culture medium for 24 hours. All HMCLs tested showed minimal changes in both MYC expression and generation of cleaved active caspase 3 when glucose is removed from the media. Removal of glutamine however diminishes MYC expression and increases cleaved active caspase 3 levels. For U266-MYC both withdrawal conditions reduce MYC protein while caspase 3 is cleaved.

Article Snippet: Quantitative transcriptome analysis using random-primed cDNA library from endogenous cell lines INA-6, U266 and the generated MYC-positive U266-CDS-MYC cells (GATC Biotech).

Techniques: Western Blot, Expressing, Transduction, Comparison, Incubation, Phospho-proteomics, Inhibition

Journal: The Journal of Cell Biology

Article Title: The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase

doi:

Figure Lengend Snippet: p193 exists in cells associated with vaults and in other subcellular fractions. Comparison of levels of p193 during biochemical fractionation of HeLa cells. (A) Cellular protein fractions, nuclear (N), supernatant S100 (S), and microsomal pellet P100 (P) were generated from HeLa extracts and resolved by SDS-PAGE (see Materials and Methods). Immunoblot analysis was carried out using affinity-purified polyclonal antibody prepared against recombinant p193 fragment. (B) The P100 fraction was further fractionated on a discontinuous sucrose gradient (20/30/40/45/50/60%), and the proteins from the gradient fractions were resolved by SDS-PAGE. Immunoblot analysis was carried out using either affinity-purified p193 polyclonal antibody (top) or affinity-purified anti-vault polyclonal antibody (bottom). Sucrose gradient fraction sources are indicated at the top. P100 represents the starting material, and the vault sample was purified from rat liver (a lighter exposure of the detected MVP in purified vaults is shown). Arrows indicate the positions of p193 and MVP.

Article Snippet: These cells were then transformed with a HeLa cell acid fusion cDNA library in the pJG7-4 expression vector (constructed by J. Gyuris, Mitotix, Cambridge, MA, and kindly provided by Dr. Roger Brent), and about 1 million transformants were plated onto dropout media lacking Trp, His, and Ura containing glucose.

Techniques: Comparison, Fractionation, Generated, SDS Page, Western Blot, Affinity Purification, Recombinant, Purification