Journal: Viruses

Article Title: The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

doi: 10.3390/v12080857

Figure Lengend Snippet: Axl and hepatocyte growth factor receptor (HGFR) RTKs cooperate during LASV entry. ( A ) HGFR surface expression in A549 and HT-1080 cells detected by FACS analysis; ( B ) HGFR surface expression upon EMD1204063 (EMD) administration. A549 cells were incubated for 1.5 h with EMD (20 µM), and then fixed and stained for HGFR detection with specific antibody; ( C ) Dose dependent entry inhibition of rLCMV/LASV GP by EMD in A549 and HT-1080 cells. Error bars represent standard deviations ( n = 3); ( D ) Phosphorylation and expression of HGFR after HGF administration. Cells were incubated with HGFR for indicated times, lysed, and tested by WB for HGFR and phosphorylated HGFR (p-HGFR). Tubulin was used as loading control. Normalized ratios were calculated from signal ratios of (HGFR/tubulin)/(p-HGFR/tubulin); ( E ) Time-of-addition assay of HGF. HGF (50 nM) or vehicle (PBS) were added to A549 cells at the indicated times relative to infection with rLCMV-VSV G or rLCMV/LASV GP . Error bars represent standard deviations ( n = 3). Asterisk (*) denotes significant differences (student’s t -test; p < 0.01); ( F ) HGFR RTK/NP colocalization. Precooled (4 °C) A549 cells were infected, for 2 h, in the cold with rLCMV/LASV GP (MOI 50 PFU/cell), and then shifted to 37 °C for indicated times. HGFR RTK was visualized in red and viral NP in green. Representative confocal images are shown. Scale bar represents 5 µm. Percentages of double puncta (NP/HGFR RTK) values obtained from the analysis of 8 randomly selected cells per condition. Error bars represent standard deviations; ( G ) DG/HGFR RTK colocalization in mock-infected and rLCMV/LASV GP -infected cells. Live A549 cells were preincubated for 2 h in the cold with mock or rLCMV/LASV GP (MOI 50 PFU/cell), and rapidly shifted to 37 °C during indicated times. DG is visualized in red and Axl RTK in green. Representative images collected by confocal microscopy are shown. Scale bar represents 5 µm. Colocalization of DG and HGFR RTK was quantified by Pearson’s coefficient in mock and infected cells after shifting to 37 °C, for 0, 2, 5, and 10 min. Error bars represent standard deviations of 10 randomly selected cells per condition. Scale bars represent 5 µm; ( H ) Dose response inhibition of rLCMV/LASV GP relative entry into A549 and HT-1080 cells by R428, alone or in combination with EMD (10 µM). Relative entry values were normalized to the infectivity in the absence of drug (DMSO). Error bars represent standard deviations ( n = 3).

Article Snippet: Purified polyclonal goat IgG anti human Axl (#AF154) and anti-human HGFR (for IFA, #AF276) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Incubation, Staining, Inhibition, Phospho-proteomics, Control, Infection, Confocal Microscopy

Journal: Viruses

Article Title: The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

doi: 10.3390/v12080857

Figure Lengend Snippet: Drug interaction (R428/EMD) analysis in HT-1080 cells. Analysis performed with CompuSyn [ 95 ]. CI values <1 means synergistic interaction, approximately 1 additive effect, and >1 antagonistic interactionAxl and HGFR participate in classical macropinocytosis.

Article Snippet: Purified polyclonal goat IgG anti human Axl (#AF154) and anti-human HGFR (for IFA, #AF276) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Viruses

Article Title: The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

doi: 10.3390/v12080857

Figure Lengend Snippet: R428 and EMD inhibit macropinocytosis-related rLCMV/LASV GP internalization. ( A ) A549 cells were pretreated with DMSO, 40 µM EIPA, 5 µM R428, or 40 µM EMD, for 30 min. Subsequently, cells were incubated with Alexa Fluor 488 labeled dextran (250 µg/mL) in the presence of the inhibitors, for 40 min, at 37 °C, washed, fixed with PFA, and subjected to confocal microscopy. Note: EIPA treatment resulted in a faint blue fluorescence throughout the cytosol. Representative fields for each condition are shown. Scale bar: 10 µm; ( B ) Precooled A549 cells were incubated for 2 h, at 4 °C, with rLCMV/LASV GP (MOI 50 PFU/cell). Cells were washed and either fixed or shifted to 37 °C, for 45 min, in the presence of DMSO or inhibitors (same concentrations as in ( A )), as indicated. Then, cells were fixed, stained for viral NP (113 antibody), and analyzed by confocal microscopy. Representative fields are shown for each condition ( n = 10). Scale bar corresponds to 10 µm. Interestingly, whereas Axl RTK inhibition by R428 decreased the number of dextran-labeled vesicles, HGFR inhibition by EMD resulted in an accumulation of vesicles in proximity to the plasma membrane ( A). In agreement with previous reports [ , ], we also found in viral infection experiments that 18.6% (±7.7%) of total Alexa Fluor 488-conjugated, dextran-containing vesicles also contained rLCMV/LASV GP viral particles .

Article Snippet: Purified polyclonal goat IgG anti human Axl (#AF154) and anti-human HGFR (for IFA, #AF276) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Labeling, Confocal Microscopy, Fluorescence, Staining, Inhibition, Clinical Proteomics, Membrane, Infection

Journal: Scientific Reports

Article Title: USP8 modulates ubiquitination of LRIG1 for Met degradation

doi: 10.1038/srep04980

Figure Lengend Snippet: (a) LRIG1 protein levels in patient-derived lung tumor xenograft samples were measured by ELISA. Each symbol represents one tumor sample. (b) USP8 protein levels were measured by immunoblot in patient-derived lung tumor xenograft samples. (c) Met protein levels in patient-derived lung tumor xenograft samples were measured by ELISA, and expressed as a percentage (%) relative to the amount of Met in vehicle-treated tumor. Asterisks (*) represent P-values according to Student's t test, *P < 0.02, **P < 0.0001. (d) Patient-derived lung tumor growth over time. Each group consists of 10 mice. Tumor volumes measured on indicated days are plotted (mean and s.e.m.) for treatment groups (SAIT301) and vehicle (negative control: PBS) group. Asterisks (*) represent P-values versus vehicle group according to repeated measures ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: For the sandwich ELISA, total Met levels in homogenized tumor lysates were determined using human total HGF R/c-Met ELISA kit (R&D systems).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control