Journal: mBio

Article Title: Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin

doi: 10.1128/mbio.01441-23

Figure Lengend Snippet: Nuclear transport blockage prevents the secretion and activity of secreted alkaline phosphatase (SEAP). ( A ) A549 cells expressing SEAP or control (mock) cells were treated for 48 h with DMSO or IVM (1 µM). Lysates and supernatants were analyzed by western blot using a SEAP-specific antibody. iSEAP and eSEAP, intra- and extracellular SEAP, respectively. ( B ) Quantification of the SEAP signal in lysates and culture supernatant of cells treated like in panel A . Values are displayed as the ratio of supernatant/lysate and normalized to those of DMSO-treated cells. N = 5 biological replicates. ( C ) Extracellular phosphatase activity released from cells treated like in panel A . Values were normalized to those of DMSO-treated cells. N = 4 biological replicates. ( D ) Intracellular phosphatase activity normalized to SEAP abundance determined by western blot of cell lysates obtained like in panel A . The ratio was normalized to values obtained for DMSO-treated cells. N = 4 biological replicates. ( E ) A549 cells expressing SEAP were transfected with KPNB1-targeting or NT siRNAs. Forty-eight hours post-transfection, the medium was changed for 48 h, and cell lysate and culture supernatant were harvested 48 h later. Samples were analyzed by western blot using a SEAP-specific antibody. ( F ) Quantification of SEAP signals in western blots as shown in panel E . N = 7 biological replicates. ( G ) Quantification of SEAP activities as in panel C . N = 8 biological replicates. ( H ) SEAP activity normalized to total intracellular SEAP amounts. Values in panels F – G were normalized to those obtained with siNT-transfected cells. N = 7 biological replicates. ( I ) Lysates of mock vs DENV-infected A549 cells were analyzed by western blot after sample loading under reducing or non-reducing conditions and detection of NS1 using conformation-specific DN3 antibody. ( J ) Lysates of A549 cells transfected with given siRNAs for 48 h and infected with DENV for 48 h (left panel) or infected with DENV and treated with IVM (1 µM) or DMSO for 48 h (right panel) were analyzed by NS1-specific western blot after sample loading under reducing or non-reducing conditions. For quantification, NS1 signal was normalized to GAPDH and the ratio of non-reducing over reducing sample signal was calculated. Values were normalized to those obtained with siNT-transfected or IVM-treated cells. Data are represented as mean ± SEM of three or six independent experiments. ( K ) NS1 in lysate (intra) and culture supernatant (extra) of mock and DENV-infected A549 cells. ( L ) Lysate (intra) and supernatant (extra) of DENV-infected A549 cells were treated with endoH or PNGase prior to NS1-specific western blot analysis. ( M ) Lysates of A549 cells transfected with given siRNAs for 48 h and infected with DENV for 48 h (top panel) or infected with DENV and treated with IVM (1 µM) or DMSO for 48 h (bottom panel) were analyzed by NS1-specific western blot. Empty and filled arrowheads point to mature and immature forms of NS1, respectively. Quantification of seven or eight independent biological replicates is shown on the right. Data are represented as mean ± SEM. Each dot in the graphs corresponds to the value of an individual experiment. In all graphs, statistical significance was determined by one-sample t -test or ratio-paired t -test for panel M. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For activity measurement, cell lysates and supernatants were analyzed using the colorimetric Secreted Alkaline Phosphatase Reporter Assay Kit (Novus Biologicals) according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Control, Western Blot, Transfection, Infection

Journal: mBio

Article Title: Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin

doi: 10.1128/mbio.01441-23

Figure Lengend Snippet: Key resource table ^a

Article Snippet: For activity measurement, cell lysates and supernatants were analyzed using the colorimetric Secreted Alkaline Phosphatase Reporter Assay Kit (Novus Biologicals) according to the manufacturer’s instructions.

Techniques: Virus, Recombinant, Cell Viability Assay, Extraction, Reverse Transcription, SYBR Green Assay, Reporter Assay, Negative Control, Software