anti-sars-cov-2 nsp5  (Proteintech)

Journal: Frontiers in Microbiology

Article Title: Quercetin, a flavonoid, combats rotavirus infection by deactivating rotavirus-induced pro-survival NF-κB pathway

doi: 10.3389/fmicb.2022.951716

Figure Lengend Snippet: Quercetin treatment diminishes rotavirus replication in vitro . (A) Chemical structure of quercetin is shown. (B) Western blot is performed from SA11(MOI 3)-infected MA104 cells (12 hpi) treated with DMSO or quercetin (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM) to evaluate the expression of viral proteins VP1 and VP6. β-actin is taken as internal loading control. (C) Protein expression of VP1, VP6, NSP1, and β-actin was checked by immunoblot from SA11-infected MA104 cells treated either DMSO or quercetin (50 μM) incubated for 0, 4, 8, and 12 hpi, respectively. (D) Immunofluorescence was performed with RV-SA11 (MOI 3)-infected MA104 cells in the presence of DMSO and quercetin (6.25, 12.5, 25, and 50 μM). At 8 hpi, cells were fixed and permeabilized followed by staining with anti-NSP5 antibody. Secondary staining was done with rhodamine-labeled anti-rabbit antibody. DAPI was used for mounting. Finally, cells were visualized with confocal microscope (63× oil immersion); scale bar 10 μm. (E) Minimum of 100 cells from different fields from each experimental slide were selected randomly and analyzed for the quantification of viroplasm-positive cells. The data were represented as mean viroplasm-positive cells (%) ± SD of three experimental replicates. (F) Plaque assay was performed from RV-SA11 (MOI 1)-infected MA104 cell treated with graded concentration of quercetin (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM) for 24 hpi to measure IC 50. Each bar represents mean ± SD of three independent experiments (Unpaired student's t-test, *** p < 0.001).

Article Snippet: Primary staining was done with anti-NSP5 antibody raised in rabbit followed by secondary staining with DyLight488-labeled goat anti-rabbit antibody (Thermo Scientific TM ).

Techniques: In Vitro, Western Blot, Infection, Expressing, Control, Incubation, Immunofluorescence, Staining, Labeling, Microscopy, Plaque Assay, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Quercetin, a flavonoid, combats rotavirus infection by deactivating rotavirus-induced pro-survival NF-κB pathway

doi: 10.3389/fmicb.2022.951716

Figure Lengend Snippet: Quercetin exhibits anti-rotaviral activity independent of viral strain: (A) Cell lysates were prepared to perform Western blot by infecting MA104 cells with RV-A513 strain followed by treatment with DMSO or increasing concentration of quercetin (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM) to assess viral protein expression. The expression of β-actin is checked as internal loading control. (B) Similarly, RV-Wa strain-infected MA104 cells are treated with DMSO or increasing doses of quercetin (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM) to check viral proteins along with β-actin as loading control. (C,D) MA104 cells were infected with (C) RV-A5-13 and (D) RV-Wa (MOI 3) in the presence of DMSO and quercetin (6.25, 12.5, 25, and 50 μM) for 6 hpi. Cells were fixed, permeabilized, and stained primarily with anti-NSP5 antibody (raised in rabbit). Next, secondary staining was done with rhodamine-labeled anti-rabbit secondary antibody. DAPI was used for mounting. Finally, cells were visualized with confocal microscope (63× oil immersion); scale bar 10 μm. Viroplasm positive cells are quantified and analyzed by taking into account minimum 100 cells selected randomly from each experimental slides. The data were represented as mean-infected cells (%) ± SD of three experimental replicates. (E,F) IC 50 was calculated from the quantification of viral particle produced by plaque assay which was performed from (E) A5-13- and (F) Wa-infected MA104 cells treated with DMSO and graded concentration of quercetin (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM), for 24 hpi. Each bar represents mean ± SD of three independent experiments (Unpaired student's t-test, *** p < 0.001).

Article Snippet: Primary staining was done with anti-NSP5 antibody raised in rabbit followed by secondary staining with DyLight488-labeled goat anti-rabbit antibody (Thermo Scientific TM ).

Techniques: Activity Assay, Western Blot, Concentration Assay, Expressing, Control, Infection, Staining, Labeling, Microscopy, Produced, Plaque Assay