Journal: Nature Communications

Article Title: A viral expression factor behaves as a prion

doi: 10.1038/s41467-018-08180-z

Figure Lengend Snippet: Characterization of the aggregates of LEF-10 and LEF-10 L21A in virus-infected Sf 9 cells. a Fluorescence microscope images displayed that wild-type LEF-10 and LEF-10 L21A fused with EGFP were driven by a tandem actin / p10 promoter (Supplementary Fig. ) and they could rescue the BacmidΔ lef-10 (more mutants in Supplementary Fig. ). b Flow cytometry analysis revealed that the expression level of LEF-10 L21A -EGFP was higher than that of LEF-10-EGFP. c Laser confocal microscopy images of over-expressed LEF-10-EGFP and LEF-10 L21A -EGFP in infected Sf 9 cells at 48 hpi (Related to Supplementary Fig. ). Both diffuse fluorescence and non-diffuse fluorescence could be observed in Sf 9 cells expressing virus-encoded LEF-10-EGFP and LEF-10 L21A -EGFP. Scale bar, 50 μm. d Percentage of cells which harbored puncta fluorescence formed by over-expressed LEF-10-EGFP or LEF-10 L21A -EGFP were examined from 30 individual fields under laser confocal microscopy images. Bars represented mean ± SD. Two-tailed unpaired Student’s t -test was performed (**** P ≤ 0.0001). Source data are provided as a Source Data file. e Laser confocal microscopy images of LEF-10-EGFP and LEF-10 L21A -EGFP under the control of native lef-10 promoter (Supplementary Fig. ) in infected Sf 9 cells at 24, 36 and 48 hpi, MOI = 10. Scale bar, 50 μm. f Percentage of cells which harbored puncta fluorescence formed by LEF-10-EGFP or LEF-10 L21A -EGFP under the control of native lef-10 promoter (Supplementary Fig. ) were examined from 30 individual fields of laser confocal microscopy images at the indicated hours post-infection. Bars represented mean ± SD. Two-tailed unpaired Student’s t -test was performed (**** P ≤ 0.0001). Source data are provided as a Source Data file. g SDD-AGE analysis of aggregates formed by LEF-10-EGFP-His and LEF-10 L21A -EGFP-His fusion proteins in infected Sf 9 cells at 48 hpi. Both chimeras were able to form such aggregates, albeit with different polymer size distribution. h Western blot analysis of aggregates formed by LEF-10-EGFP-His and LEF-10 L21A -EGFP-His fusion proteins in infected Sf 9 cells at 48 hpi. The high molecular-weight fraction remained in the stacking gel, and the low molecular-weight fraction and monomers were detected in the resolving gel. The LEF-10-EGFP-His and LEF-10 L21A -EGFP-His proteins in g and h were detected using α-His antibody

Article Snippet: Sf 9 cells were infected with recombinant baculovirus vAc/ P actin-p10 -LEF-10-EGFP and its 10 variants (Supplementary Table ) were observed at 48 hpi with Revolve R4 fluorescence microscopy (Echo Laboratories) at an emission wavelength of 530 nm (Supplementary Fig. ).

Techniques: Virus, Infection, Fluorescence, Microscopy, Flow Cytometry, Expressing, Confocal Microscopy, Two Tailed Test, Control, Polymer, Western Blot, High Molecular Weight, Molecular Weight