Figure 1. Imaging translation of single mRNAs with the SunTag system. (A) Schematic of the Suntagx56-Ki67 reporter mRNA. (B) Effect of puromycin (Puro) on the brighter protein foci labeled by the scFv-sfGFP. Panels represent microscopy images of HeLa cells stably expressing the scFv-sfGFP and the SunTagx56-Ki67 mRNA and untreated (left) or treated with puromycin (right). The blue arrow indicates a spot corresponding to a single molecule of SunTagx56-Ki67 protein, whereas the red arrow points to a brighter protein foci. (bottom right inset) A zoom of the boxed area (10 × 10 µm). (top left inset) An additional zoom of the first inset to show single molecules of proteins. Bar, 10 µm. (C) Expression of the scFV-sfGFP alone does not generate foci but a homogenous signal. The microscopy image is taken from HeLa cells stably transduced with the scFv retroviral vector (the parental cells of the clone shown in B). Bar, 10 µm. (D) Quantification of the number of brighter protein foci per cell (±2 SD; mean of four experiments; >160 cells counted in each condition). (E) Colocalization of the bright protein foci labeled by the scFv-sfGFP, with the SunTagx56-Ki67 mRNAs. Panels represent microscopy images of cells stably expressing the scFv-sfGFP and the SunTagx56-Ki67 mRNA and labeled with smFISH probes recognizing the SunTag-Hygro sequences. Right panel: color overlay of the smFISH image (red, left panel) and the SunTag signal (green, middle panel). The blue arrow indicates a spot corresponding to a single molecule of SunTagx56-Ki67 protein (middle panel), whereas the red arrow points to a brighter protein foci that colocalizes with the SunTagx56-Ki67 mRNA. Inset: a zoom of the boxed area (4 × 4 µm). Bar, 4 µm.
Figure 1.

Imaging translation of single mRNAs with the SunTag system. (A) Schematic of the Suntagx56-Ki67 reporter mRNA. (B) Effect of puromycin (Puro) on the brighter protein foci labeled by the scFv-sfGFP. Panels represent microscopy images of HeLa cells stably expressing the scFv-sfGFP and the SunTagx56-Ki67 mRNA and untreated (left) or treated with puromycin (right). The blue arrow indicates a spot corresponding to a single molecule of SunTagx56-Ki67 protein, whereas the red arrow points to a brighter protein foci. (bottom right inset) A zoom of the boxed area (10 × 10 µm). (top left inset) An additional zoom of the first inset to show single molecules of proteins. Bar, 10 µm. (C) Expression of the scFV-sfGFP alone does not generate foci but a homogenous signal. The microscopy image is taken from HeLa cells stably transduced with the scFv retroviral vector (the parental cells of the clone shown in B). Bar, 10 µm. (D) Quantification of the number of brighter protein foci per cell (±2 SD; mean of four experiments; >160 cells counted in each condition). (E) Colocalization of the bright protein foci labeled by the scFv-sfGFP, with the SunTagx56-Ki67 mRNAs. Panels represent microscopy images of cells stably expressing the scFv-sfGFP and the SunTagx56-Ki67 mRNA and labeled with smFISH probes recognizing the SunTag-Hygro sequences. Right panel: color overlay of the smFISH image (red, left panel) and the SunTag signal (green, middle panel). The blue arrow indicates a spot corresponding to a single molecule of SunTagx56-Ki67 protein (middle panel), whereas the red arrow points to a brighter protein foci that colocalizes with the SunTagx56-Ki67 mRNA. Inset: a zoom of the boxed area (4 × 4 µm). Bar, 4 µm.

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