Journal: Nature Communications

Article Title: Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation

doi: 10.1038/s41467-024-50437-3

Figure Lengend Snippet: a Schematic representation of the workflow to perform click-labeling and immunofluorescence staining on fixed cells that were treated with one of the PAL enantiomers or DMSO control (vehicle). Note that a click reaction with Picolyl-azide-Cy5.5 was performed regardless of treatment condition. b Example confocal microscopic images (60x). Cells were transfected with the pIRO-D WT construct encoding the NS1-5 polyprotein or mock transfected and treated with either 1 µM Compound A, 1 µM Compound B or vehicle. Shown are the PAL fluorescence signal, the IF signals of NS3 and NS4B and a merge that includes nuclear DNA stain with DAPI. Scale bar = 10 µm. c For each sample transfected with pIRO-D WT, 21 cell profiles (7 from each of the 3 independent experiments) were examined for Pearson correlation coefficients between the PAL and the NS4B signal. Shown is the mean with standard deviation (SD). A one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical parameters. d Specificity of Compound A colocalization with NS4B was determined using cells transfected with a ZIKV polyprotein encoding construct (pIRO-Z). Example images obtained as described for b are shown. Scalebar = 10 µm. e Quantification of PAL and ZIKV NS4B signal overlap as described for c . Per sample, 21 cell profiles (7 from each of the 3 independent experiments) were analyzed and plotted with mean and SD. The indicated p -value resulted from an unpaired two-tailed t -test. f NS4B mutations conferring various degrees of resistance against JNJ-A07 were examined for their impact on colocalization between NS4B and PAL signals. The corresponding IF panel is shown in Supplementary Fig. . Pearson correlation coefficients of a total of 21 cell profiles are plotted (7 each from three independent experiments). Statistical significance was calculated using a two-way ANOVA test and Šidák’s multiple comparisons. ns = non-significant.

Article Snippet: After a brief wash in PBS, coverslips were placed upside down on 20 µl drops of click-reaction mixture (aqueous solution with 1 mM ascorbic acid, 1 mM CuSO 4 ·5 H 2 O, 100 µM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 50 µM Picolyl-Azide-Cy5.5 [Jena Bioscience]) in a humid, dark chamber.

Techniques: Labeling, Immunofluorescence, Staining, Control, Transfection, Construct, Fluorescence, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation

doi: 10.1038/s41467-024-50437-3

Figure Lengend Snippet: a Schematic representation of the workflow. Note that although cells were treated with either Compound A or DMSO control (vehicle), the click reaction was performed on the beads with Picolyl-azide-Cy5.5 azide in both cases. Also note that different subclones of Huh7/Lunet-T7 cells were used: naïve ones in which 2K-NS4B-HA or NS4A-2K-NS4B-HA were transiently expressed and a subclone stably expressing Calnexin-HA. b Representative western blot showing the input controls. Due to its stable expression, the signal of Calnexin-HA is stronger than the ones of the transiently expressed viral proteins. Mock samples were derived from Huh7/Lunet-T7 NS2B-NS3 cells treated with transfection reagent only. c Representative images to analyze compound binding to individual proteins. Pull-down samples were loaded onto a gel, which was analyzed for Cy5.5 fluorescence signal using a LICOR Odyssey imaging system (middle plane). Thereafter, proteins were analyzed by HA-specific western blot (top panel). An overlay of both images is shown at the bottom. Mock transfected samples in lanes 1 and 2 reflect the unspecific background in the absence of HA-tagged proteins. For each construct, there is a Compound A-treated sample and a vehicle-treated sample to distinguish between non-specific labeling and specific binding to Compound A. Owing to high expression level, Calnexin-HA samples were diluted 1:500 prior to loading to reach western blot signal intensities in a linear range comparable with the other samples. d Quantification of compound binding efficiency; shown is the ratio of the fluorescence signals (reflecting Compound A bound proteins) to the corresponding HA western blot signal (reflecting the target protein). Non-specific compound labelling was subtracted by using the analogous ratio of the individual vehicle control. Bar graph shows individual data points, as well as mean and standard error of the mean (SEM; n = 4). Indicated p -values were calculated using one-way ANOVA with subsequent Tukey’s multiple comparisons test.

Article Snippet: After a brief wash in PBS, coverslips were placed upside down on 20 µl drops of click-reaction mixture (aqueous solution with 1 mM ascorbic acid, 1 mM CuSO 4 ·5 H 2 O, 100 µM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 50 µM Picolyl-Azide-Cy5.5 [Jena Bioscience]) in a humid, dark chamber.

Techniques: Control, Stable Transfection, Expressing, Western Blot, Derivative Assay, Transfection, Binding Assay, Fluorescence, Imaging, Construct, Labeling