Journal: PLoS ONE

Article Title: Importin α3/Qip1 Is Involved in Multiplication of Mutant Influenza Virus with Alanine Mutation at Amino Acid 9 Independently of Nuclear Transport Function

doi: 10.1371/journal.pone.0055765

Figure Lengend Snippet: (A) Schematic diagram and subcellular localization of the N-terminal 110-amino acid region (NP110aa) NLS mutants. The unconventional NLS (aa 3-TKGTKRSYEQM-13), overlapping bipartite NLS (aa 90-KKTGGPIYRRVDGKWRRELILYDKEEIRRIWR-121) or bipartite NLS (aa 198-KRGINDRNFWRGENGRKTR-216) are indicated in gray in the schematic showing of full length NP and NP110aa. A549 cells were transfected with plasmids expressing WT and mutant NP110aas. The nuclear and cytoplasmic localization of WT and mutant NP110aas were determined by monitoring mRFP fluorescence. Over five hundred mRFP-expressing cells were counted and classified as nuclear or cytoplasmic localization. (B) Fluorescence microscope images showing mRFP, WT NP110aa and a mutant lacking the N-terminal 13-amino acid tail region (NP14-110aa) (upper panel). Nucleus is stained with DAPI (lower panel). (C) Fluorescence microscope images showing WT and mutant NP110aas. The localization of NP was consistent with that observed using mRFP fluorescence.

Article Snippet: Madin-Darby canine kidney (MDCK) cells , human lung carcinoma A549 cells (ATCC, cat no: CCL-185), African green monkey kidney COS-7 cells , human embryonic kidney HEK-293 cells (ATCC, cat no: CRL-1573) and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing Pen Strep Glutamine (PSG, Gibco) and 10% fetal bovine serum (Sigma).

Techniques: Transfection, Expressing, Mutagenesis, Fluorescence, Microscopy, Staining

Journal: PLoS ONE

Article Title: Importin α3/Qip1 Is Involved in Multiplication of Mutant Influenza Virus with Alanine Mutation at Amino Acid 9 Independently of Nuclear Transport Function

doi: 10.1371/journal.pone.0055765

Figure Lengend Snippet: (A) A549 cells were infected with the S9A NP mutant and WT NP viruses at a multiplicity of infection of 1. At 24 h post-infection, the supernatants were harvested and the virus titer was determined using plaque assays. Data represent the relative growth of the S9A mutant compared with that of the WT. (B) Mini-genome assay using the S9A NP mutant. The effect of the S9A mutation on viral transcription was determined using a mini-genome assay with the plasmid encoding the S9A NP mutant. Luciferase activity was measured 48 h after the transfection of viral protein- (PB2, PB1, PA or NP) and vNP-luc-expressing plasmids. To analyze the effect of the S9A mutation on vRNA transcription, luciferase activity was compared with that generated by the WT. Data represent the mean ± SD of measurements from three samples in each of two independent experiments (*p<0.05, by students t -test).

Article Snippet: Madin-Darby canine kidney (MDCK) cells , human lung carcinoma A549 cells (ATCC, cat no: CCL-185), African green monkey kidney COS-7 cells , human embryonic kidney HEK-293 cells (ATCC, cat no: CRL-1573) and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing Pen Strep Glutamine (PSG, Gibco) and 10% fetal bovine serum (Sigma).

Techniques: Infection, Mutagenesis, Virus, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Generated

Journal: PLoS ONE

Article Title: Importin α3/Qip1 Is Involved in Multiplication of Mutant Influenza Virus with Alanine Mutation at Amino Acid 9 Independently of Nuclear Transport Function

doi: 10.1371/journal.pone.0055765

Figure Lengend Snippet: (A) HEK-293 cells were transfected with Qip1 siRNA for 24 h. Silencing of Qip1 was confirmed by Western blotting with an antibody specific for Qip1. (B) Qip1-silenced HEK-293 cells were transfected with plasmids encoding PB2, PB1, PA, WT or S9A NP and vNP-luc, and luciferase activity was measured after 48 h. Columns and error bars represent the mean ± SD of measurements from three samples in each of three independent experiments (*p<0.05, by students t -test). (C) A549 cells were transfected with Qip1 siRNA for 48 h. Silencing of Qip1 was confirmed by Western blotting with an antibody specific against Qip1. (D) Qip1-silenced A549 cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT or S9A NP110aa. The localization of NP was consistent with that observed using mRFP fluorescence. (E) The Qip1-silenced A549 cells were infected with A/WSN/33 virus containing WT or S9A NP at an MOI of 0.01. The viral titer in supernatant of non-targeting siRNA transfected (triangle) or Qip1 siRNA transfected (square) cells was determined using a plaque assay. Data represent the mean ± SD of measurements from three samples in each of three independent experiments (*p<0.05, by students t -test).

Article Snippet: Madin-Darby canine kidney (MDCK) cells , human lung carcinoma A549 cells (ATCC, cat no: CCL-185), African green monkey kidney COS-7 cells , human embryonic kidney HEK-293 cells (ATCC, cat no: CRL-1573) and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing Pen Strep Glutamine (PSG, Gibco) and 10% fetal bovine serum (Sigma).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay, Fluorescence, Infection, Virus, Plaque Assay

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: Fluorescence microscopy images of B16 cells grown 48 h on ( A ) biocompatible glass and ( B ) gold surface; scale bars: 100 µm.

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: Scanning electron microscopy images of B16 cells grown 24 h on gold surface obtained at different magnifications and using signals from different detectors (SE2 and InLens): ( A ) 300×; ( B ) and ( C ) 500×.

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Electron Microscopy

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: Normalized values to control cells (cells not irradiated) of ( A ) mitochondrial superoxide and ( B ) ROS recorded for B16 cells at 2, 4 and 24 h after proton irradiation at dose rate of 1 Gy/min. Data are reported as mean ± SD. *p < 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Control, Irradiation

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: Cyclic voltammograms recorded at gold electrode for (grey line) culture medium and (black line) B16 cells ( A ) before and ( B ) after proton irradiation at a dose of 1 Gy.

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Irradiation

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: Cyclic voltammograms results recorded at gold working electrodes after 30 min incubation with different concentrations of H 2 O 2 for ( A ) culture medium with the corresponding ( B ) plot of oxidation current vs . H 2 O 2 concentration and ( C ) B16 cells grown at the electrode surfaces.

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Incubation, Concentration Assay

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: ( A ) Fluorescence microscopy and ( B ) FESEM images obtained for B16 cells at gold surface after interaction with H 2 O 2 ; scale bars: 100 µm.

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

doi: 10.1038/s41598-022-06277-6

Figure Lengend Snippet: OCP recorded at gold electrodes for culture medium and B16 cells before and after incubation with H 2 O 2 and proton irradiation (1 Gy at 1 Gy/min).

Article Snippet: For in vitro studies we used B16 melanoma cell line (cat. Nr. CRL-6475), ATCC (USA).

Techniques: Incubation, Irradiation