Supplementary Figure 8: Comparison of Peppers’ and MS2 system’s Performance for RNA imaging in COS-7 Cells.

(a) Schematic representation of constructs expressing NLS-tdMCP-FPs, or mRNAs fused with 4P4M dual tag. mRNAs without 4P4M tag were used as controls. (b) and (c) Fluorescence imaging of Pepper620 (red) and tdMCP-EGFP (green) in cells expressing BFP mRNA (b) or ACTB mRNA (c), with or without 4P4M tag. (d) and (e) Fluorescence imaging of Pepper530 (green) and tdMCP-mCherry (red) in cells expressing BFP mRNA (d) or ACTB mRNA (e), with or without 4P4M tag. (f-i) Quantification of the fluorescence of MS2 system channel (tdMCP-EGFP or tdMCP-mCherry) and Pepper channel (Pepper530 or Pepper620) in the cytosol of cells expressing mRNAs with or without 4P4M tag. These data showed much higher signal/background contrast was detected in the Pepper channel comparing to the MS2 system channel. Statistical comparison was performed by two-tailed t test. Data represent the mean ± s.d. (N = 50 cells). (j) Schematic representation of constructs for the targeting of ACTB mRNA to outer membrane of mitochondria through TOMM20-tdPCP-BFP tethering. (k) and (m) Fluorescence imaging of tdMCP-EGFP and Pepper530 (k) or tdMCP-mCherry and Pepper620 (m) in cells expressing mitochondrial localized ACTB mRNA with 4P4M tag. Cells were co-transfected with ACTB-4P4M-4xPP7, TOMM20-tdPCP-BFP and NLS-tdMCP-EGFP (k) or NLS-tdMCP-mCherry (m) plasmids. The fluorescence signals of the MS2 system (EGFP or mCherry) were measured in cells before HBC ligands incubation. The fluorescence signals of Pepper were measured by the enhanced fluorescence in the same cells after incubation with HBC ligands, which reflected the formation of Pepper complex spectral matched to FPs of MS2 system (Pepper530 for EGFP, Pepper620 for mCherry). (l) and (n) Quantification of the MS2 system’s or Pepper’s fluorescence signal of ACTB mRNA tagged with 4P4M tag, and TOMM20-tdPCP-BFP fluorescence in mitochondria and cytosol of the cells. Statistical comparison was performed by two-tailed t test. N.S., no significant difference. Data represent the mean ± s.d. (N = 50 cells). Scale bar, 10 µm. For b-e, k and m, at least two independent experiments were carried out with similar results.