Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: A . RPMI-8226, ALMC-2, MM.1S, and U266 cells were incubated for 48 hours in the presence or absence of lovastatin ( Lov , 2-50 μM) or DGBP (2-50 μM). Immunoblot analysis of p62, Atg3 and β-tubulin (loading control) is shown. The relative intensity ( Rel Int) values underneath the p62 and Atg3 blots represent relative levels as determined by densitometric analysis of Atg3 or p62 (relative to tubulin) of treated cells compared to control cells. The gels are representative of 3-4 independent experiments. B . ALMC-2 cells were incubated for 48 hours in the presence of 10 μM lovastatin ( Lov ) and/or mevalonate ( Mev , 5 mM), FPP (10 μM), or GGPP (10 μM) for 48 hrs. Densitometric analysis of p62 or Atg3 levels (normalized to β-tubulin levels) for the combination treatments normalized to untreated cells (control) is shown. Data are displayed as average +/− standard deviation (n=3 independent experiments). * denotes p-value <0.05 from two-sided t-testing. C . RPMI-8226 and ALMC-2 cells were incubated for 48 hours in the presence or absence of 10 μM lovastatin ( Lov ), 10 μM DGBP, 10 μM GGTI-2133 ( GGTI ), 10 mM 3-PEHPC ( 3P ), or the combination of 10 μM GGTI-2133 and 10 mM 3-PEHPC ( GGTI + 3P ). Immunoblot analysis of p62, Atg3 and β-tubulin (loading control) is shown. The gels are representative of 3-4 independent experiments. D . RPMI-8226 and ALMC-2 cells were incubated for 48 hours in the presence or absence of 10 μM lovastatin ( Lov ), 10 μM DGBP, 10 mM 3-PEHPC ( 3P ), or 5 nM bafilomycin A1 ( Baf ) with or without protease inhibitors (10 μg/mL E64d and pepstatin A, prot ). Immunoblot analysis of LC3 and β-tubulin (loading control) is shown. The gels are representative of 3 independent experiments. E . Intracellular lambda light chain levels were measured via ELISA. Data are expressed as a percentage of control (mean +/− standard deviation of 3 independent experiments). Two-sided t-testing was performed to compare the IBP inhibitor alone vs. in combination with the protease inhibitors. No significant differences between the treatment groups were observed.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Incubation, Western Blot, Control, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: RPMI-8226 cells were incubated with 2 nM bafilomycinA1, 10 μM lovastatin, or 5 mM 3-PEHPC for 48 hours. Staining was performed as described in Materials and Methods, using antibodies directed against lambda light chain (red) and PDI as an ER marker (green) in A . and RCAS1 as a Golgi marker (green) in B . DAPI was used for nuclear staining (blue). Colocalization was determined using the RGB plot profile tool in ImageJ and indicated by similarity of the patterns of red and green peaks. Scale bar 10μm.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Incubation, Staining, Marker

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: A . RPMI-8226 cells were incubated with 2 nM bafilomycin A1 ( Baf ), 10 μM lovastatin ( Lov ), or 5 mM 3-PEHPC ( 3P ) for 48 h or 10 nM bortezomib for 24 h. Staining was performed as described in Materials and Methods, using Proteostat (Enzo Life Sciences, Inc.) (green) to visualize aggresomes and an antibody directed against lambda light chain (red). DAPI was used for nuclear staining (blue). Colocalization was determined using the RGB plot profile tool in ImageJ. Scale bar 10 μm. B . The average number of aggresomes per cell was quantified using the GFP-LC3 macro for ImageJ (mean ± SEM). **** denotes P <0.0001 from unpaired two-tailed t- test comparing treated with control cells.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Incubation, Staining, Two Tailed Test, Control

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: U266 cells were treated with 2 nM bafilomycin A1 ( Baf ), 10 μM lovastatin ( Lov ) or 10 mM 3-PEHPC for 48 hours before processing for staining with LC3. A . The average number of LC3 puncta per cell and B . the average size of each punctate structure in pixels was quantified using the GFP-LC3 macro for ImageJ (mean ± SEM) (n>175). The **** denotes p <0.0001 from unpaired two-tailed t- test comparing treated with control cells. C . Representative confocal maximum projections are shown for each treatment with LC3 in green and DAPI staining the nucleus in blue. D . RPMI-8226 cells were incubated with 2 nM bafilomycin A1, 10μM lovastatin, 5mM 3-PEHPC or a co-treatment with bafilomycin A1 ( Baf ) and lovastatin ( Lov ) for 48 hours. Staining was performed as described in Materials and Methods, using CytoID (Enzo Life Sciences, Inc., Farmingdale, NY) to visualize autophagosomes and an antibody directed against lambda light chain. DAPI was used for nuclear staining. Colocalization was determined using the RGB plot profile tool in ImageJ. Scale bar 10μm.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Staining, Two Tailed Test, Control, Incubation

Journal: Blood Cancer Journal

Article Title: Downregulation of PA28α induces proteasome remodeling and results in resistance to proteasome inhibitors in multiple myeloma

doi: 10.1038/s41408-020-00393-0

Figure Lengend Snippet: a Western blot analysis of ubiquitinated proteins in total protein lysates extracted from LP1 and RPMI8226 PA28α knockdown stable cells. β-actin as a loading control. b OPP pulse-chase assay of proteasome degradation and protein synthesis in LP1 PA28α knockdown stable cells. Western blot analysis of immunoglobulin lambda light chain (ƛ IgL) ( c ), eIF2α, p62/SQSTM1, and LC3B ( d ) in LP1 and RPMI8226 PA28α knockdown stable cells, β-actin as a loading control. ** P < 0.01, Student’s t test. e Working model of PA28α knockdown in MM.

Article Snippet: Antibodies used were as follows: PA28α (Cell Signaling), PSMA2 (Cell Signaling), S5a (Cell signaling), PA28β (Cell Signaling), Phospho-eIF2α (Ser51) (Cell signaling), eIF2α (Cell signaling), α-tubulin (Genetex), PA28γ (Genetex), PSMB5 (Genetex), PSMB6 (Enzo life science), PSMB7 (Genetex), PSMB8 (Genetex), PSMB9 (R&D systems), PSMB10 (R&D systems), Rpt5 (Enzo life science), Ubiquitin (Cell Signaling), β-actin (Santa Cruz Technology), TCF11/NRF1 (Cell Signaling), LC3B (Cell Signaling), p62/SQSTM1 (MBL International) human Ig lambda light chain (R&D systems), actin (Sigma). pLKO.1 empty vector, shRNA vector targeting human PA28α, NRF1 siRNA (ON-TARGETplus SMARTpool), PA28α siRNA (Accell SMARTpool), and control siRNA were purchased from Dharmacon.

Techniques: Western Blot, Knockdown, Control, Pulse Chase

Journal: Cells

Article Title: Empagliflozin Preserves Cardiomyocyte Structural Homeostasis via the Stabilization of the Integrin α5–Desmocollin-2 Adhesion Axis in Sepsis-Induced Cardiomyopathy

doi: 10.3390/cells14181452

Figure Lengend Snippet: Empagliflozin ameliorates LPS-induced cardiac dysfunction and myocardial injury. ( A ) Echocardiography panels: top, B-mode long-axis image; middle, M-mode from the parasternal short-axis at the papillary muscle level; bottom, pulsed-wave Doppler of transmitral inflow (E and A waves). ( B ) Quantification of cardiac function parameters: Ejection Fraction (EF), Fractional Shortening (FS), and E/A Ratio. ( C ) Representative images of Hematoxylin and Eosin (H & E) staining of myocardial tissue sections showing inflammatory infiltration. Longitudinal and cross sections are shown with a scale bar of 1 mm. Magnified views of perivascular and interstitial regions are shown with a scale bar of 20 µm; green arrows indicate inflammatory cells. ( D ) Representative images of Masson’s trichrome staining revealing myocardial fibrosis. Longitudinal sections are shown with a scale bar of 1 mm, and cross sections with a scale bar of 0.5 mm. Magnified views of perivascular and interstitial regions are shown with a scale bar of 40 µm; yellow arrows indicate collagen deposition (fibrotic areas). ( E ) Quantification of the fibrotic area from Masson’s trichrome staining. ( F ) Serum concentrations of cardiac troponin T (cTnT) and C-reactive protein (CRP) measured by ELISA. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. **** p < 0.0001, *** p < 0.001, ** p < 0.01.

Article Snippet: Serum levels of cardiac troponin T (cTnT) and C-reactive protein (CRP) were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (cTnT: Cat. No. orb565454, Biorbyt, Wuhan, China; CRP: Cat. No. orb219654, Biorbyt, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: Schematic representation of the recombinant measles virus (MV) strains used in the experiments: MV-lambda, MV-lambda-NAP, MV-s-NAP and MV-NIS. NAP, neutrophil-activating protein.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: Recombinant, Virus

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: In vitro antitumor activity of engineered measles virus (MV) strains against breast cancer cells. (a) MCF-7 cell line and the highly tumorigenic MDA231-lu-P4 in vivo derivative of (b) MDA-MB-231 cells were infected with MV strains at an multiplicity of infection (MOI) = 1.0 and cell viability was determined by MTT assay. Both MV-lambda-NAP and MV-lambda viruses propagated rapidly causing complete destruction of the breast cancer monolayers 72 hours postinfection. MV-s-NAP and MV-NIS showed similar tumor cell-killing kinetics with complete eradication of MCF-7 cells or ~80% reduction of cell viability of MDA231-lu-P4 cells. (c) Vero cells were used as control. The data are presented as percent of untreated control cells ± SD. (d) NAP transgene induced significant interleukin (IL)-8 expression in MV vector-infected THP-1 cells. THP-1 monocytic cells were infected with MV-s-NAP or control MV-lambda virus at a MOI = 0.1 and incubated for 72 hours. Supernatants were collected and IL-8 concentration was measured by enzyme-linked immunosorbent assay (ELISA) specific for human IL-8. The effect of TNF-α (one of the known NAP-triggered inflammatory cytokines) on proliferation of MV-infected or uninfected MDA231-lu-P4 cells was examined by MTT assay (e,f). MDA231-lu-P4 cells were plated at 104 cell/well density and inoculated at MOI = 0.5 of MV-s-NAP in the presence or absence of 250 U/ml recombinant human TNF-α (e). The same experiment was repeated with lower cell density (2.5 × 103 per well) and MV-s-NAP at MOI of 0.25 (f).

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: In Vitro, Activity Assay, Virus, In Vivo, Infection, MTT Assay, Control, Expressing, Plasmid Preparation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: Measles virus (MV) infection and neutrophil-activating protein (NAP) transgene expression in the malignant pleural effusion of mice-bearing MDA231-lu-P4 pleural xenografts. Mice were treated by a single transthoracic (t.t.). injection of MV-s-NAP or MV-lambda. NAP transgene expression in pleural fluid was demonstrated by immunoblotting using (a) NAP-specific monoclonal antibody (MAb) 16F4 or (b) lambda chain-specific antibody. The secretory form of the NAP transgene was detected in the pleural fluid of four of four MV-s-NAP-treated mice (lanes 1–4) but not in MV-lambda-injected (lanes 5, 6) control mice (a). Human lambda light chain was detected in three of four samples from MV-lambda-injected (lanes 3–6) mice but not in samples from MV-s-NAP-treated (lanes 1, 2) animals (b) using a lambda chain-specific detection antibody. MV-s-NAP (c) induced large multinucleated syncytia in infected tumor cells (Giemsa staining) in the pleural fluid of mice with MDA231-lu-P4 xenografts. Immunohistochemistry (IHC) staining for neutrophils in the pleural fluid of MV-s-NAP is shown in (d). MV-s-NAP was isolated from the pleural fluid by overlay on Vero cells. MV-s-NAP induced giant syncytia formation 24 hours after overlay (e). Uninfected control Vero cells (f).

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: Virus, Infection, Expressing, Injection, Western Blot, Control, Staining, Immunohistochemistry, Isolation

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: Therapeutic effect of neutrophil-activating protein (NAP)-expressing measles virus (MV) strains against lung metastatic breast cancer compared to control MV-NIS. (a) Engraftment of the systemically injected MDA231-lu-P3 or P4 cells was confirmed by bioluminescence imaging. (b,c) The animals with MDA231-lu-P3 lung tumors (8 per group) were treated with 10 repeat intravenous (i.v.) injections of 2 × 106 TCID50 of MV-lambda-NAP or heat-inactivated (HI-control) control. In a separate in vivo experiment MDA231-lu-P4 lung metastatic xenografts (9 mice per group) were treated by 7 i.v. injections of 106 TCID50 of MV-s-NAP, MV-NIS or the corresponding heat-inactivated controls (d–f). Both MV-lambda-NAP and MV-s-NAP improved the median survival with 10–12 days (P < 0.05) in this aggressive model of breast cancer metastatic to the lungs.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: Expressing, Virus, Control, Injection, Imaging, In Vivo

Journal: Bmc Cancer [Electronic Resource]

Article Title: Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe

doi: 10.1186/1471-2407-6-6

Figure Lengend Snippet: Antibody source and usage

Article Snippet: CREST serum against human kinetochore proteins (Acris Antibodies, Hiddenhausen, Germany) , Mouse anti-human IgG-FITC (Dr. MS Cragg).

Techniques: Plasmid Preparation