Fig. 3: WM-smFISH enables spatial and quantitative characterization of gene expression at RNA and protein levels upon exogenous stimulus.

a, Representative images for the detection of VENUS mRNA (magenta) and protein (green) in pDR5rev::3xVENUS-N7 reporter seedlings treated with dimethylsulfoxide, NAA 1 µM, or NAA 10 µM for 2 h. The contours of cells were visualized with Renaissance 2200 dye (white). Scale bars, 20 μm. b,c, Heatmaps representing the levels of the mean signal intensity per cell detected in the channels for RNA (b) or protein (c) detection in the representative images shown in a. d, Heatmaps representing the ratio between the RNA and protein signal intensities per cell in the representative images shown in a. e,f, Density plots showing the distributions for the number of transcripts (e) and total protein intensity (f) per cell detected in all the treated roots (n for dimethylsulfoxide = 1,659 cells examined over 10 roots, n for NAA 1 µM = 1,830 cells examined over 10 roots, n for NAA 10 µM = 1,456 cells examined over 9 roots). The dashed lines represent the mean values for each condition. Violin plots showing the log-normalized (base e) distributions. Boxes inside show the interquartile range (IQR; 25%–75%), indicating the median values as a horizontal line. Whiskers show the ±1.58× IQR value. The P values for analysis of variance followed by one-sided Tukey’s HSD tests are shown in the inserts: dimethylsulfoxide versus NAA 1 µM (P < 0.0001), dimethylsulfoxide versus NAA 10 µM (P < 0.001), NAA 1 µM versus NAA 10 µM (P = 0.307) (e); dimethylsulfoxide versus NAA 1 µM (P < 0.0001), dimethylsulfoxide versus NAA 10 µM (P < 0.001), NAA 1 µM versus NAA 10 µM (P < 0.0001) (f). Experiments were repeated independently two times.