Fig. 1: Uptake, intracellular distribution of G12 and G12(S) added directly to the culture medium.

Images of the TAMRA-G12, TAMRA-G12(S), and nuclei (Hoechst 33342) in A549 cells were observed at the times indicated below and are shown in green (TAMRA) and blue (Hoechst 33342) pseudocolors, respectively (a–e). a Cellular uptake of TAMRA-G12 (2 μM, 30 min). b Uptake of TAMRA-G12(S) (0.2 μM) in the absence or presence of a 10-fold concentration of unlabeled G12(S) (2 μM) for 1 h. c Time-dependent incorporation of TAMRA-G12(S) signals from 0 to 15 min after the addition of 2 μM TAMRA-G12(S). d FRAP analysis of TAMRA-G12(S). Fluorescence was bleached for 5 min at 100% laser power. The photobleached area is marked with a white circle in the leftmost image. Images were acquired over time for the times (min) shown in the figure. e Distribution of TAMRA-G12(S) (green) and ER-Tracker Red (red). Cells were treated with 2 μM TAMRA-G12(S) in the medium for 1 h and further incubated with 1 μM ER-Tracker Red and NucBlue (Hoechst 33342). Scale bars are shown in (a–e). f Cytotoxicity of G12(S) on A549. The cell viabilities were examined after 24 h of culture in the presence of 0–10 μM G12(S) using AlamerBlue. One-way analysis of variance (ANOVA) was used for the statistical analysis (n = 3, P-values = 0.3582, F-value = 1.230). There is no significant difference between the combinations. g Effect of G12(S) on protein synthesis in A549 were determined using the puromycin uptake assay SuNSET method²¹. Cells were cultured as a condition of the virus infection in the presence of 1 and 2 μM G12(S) for 24 h. Cycloheximide (10 μg/mL CHM) instead of G12(S) was used to inhibit protein synthesis as a control. See Supplementary Fig. 1f for western blotting data for the SuNSET. Reporter gene assays for IFN-β promoter (h) and a promoter containing the NFκB binding sites (i) in HEK293T cells. The values shown are relative values when the control (without G12(S) and poly I:C) is set as “1”. The reporter genes activated by polyI:C were not induced by 2 μM G12(S), but rather suppressed. Statistical analysis of the significance for (g–i) were carried out by One-Way ANOVA (g n = 3, F(3,8) = 43.26, P < 0.0001; h n = 3, F(3, 8) = 67.30, P < 0.0001; i n = 3, F (3, 8) = 290.1, P < 0.0001), and Dunnett’s multiple comparison test (g) or Tukey’s multiple comparison test (h, i). Data are shown by Box-and-whisker plot are used with P-values for the multiple comparison (f–i).