Extended Data Fig. 4: Confocal images and spot analysis comparing 1xmNG-dMCP to 1xmNG-NLS-tdMCP.
(a) Maximum intensity projection confocal microscopy images of fixed U2OS cells expressing H2B-mCherry-24xMS2-pA RNA tagged by 1xmNG-HA-dMCP (top two rows, images D1-D10) or 1xmNG-HA-NLS-tdMCP (bottom two rows, images N1-N10). Images are displayed with signal inverted to better preserve visibility of spots after image compression below original 2046-pixel width. (b) Zoomed images displaying regions outlined by boxes in (a). These images more accurately display the intensity and size of spots as they appear in the original images. (c,d) Signal-to-Noise Ratio (SNR) of spots in all displayed (c) dMCP and (d) NLS-MCP cells. SNR is measured as the Mean Fluorescent Intensity (MFI) of pixels within a spot divided by the mean fluorescent intensity of the entire cytosolic region excluding spots and the nucleus (see Supplementary Fig. 6 for a portrayal of how this region is defined). Both values are calculated with the average background intensity of the local culture medium (calculated as the MFI of a large boxed region in each image in which no cells were detected) subtracted. Bars represent the mean of each population. (e,f) Area in square micrometers of (e) dMCP and (f) NLS-MCP spots. Bars represent the mean of each population. (g,h) MFI of (e) dMCP and (f) NLS-MCP spots. Bars represent the mean of each population. (i) Comparison of mean SNR between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: *** P = 0.0002. (j) Comparison of mean spot area between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ns P = 0.1073. (k) Comparison of mean spot MFI between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ns P = 0.7412. (l) Comparison of MFI for cytosol (as described above) between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ****P = 8.9 × 10−5. (m) Comparison of noise- calculated cytosol MFI minus average background intensities of local culture medium (as described above) between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ** P = 0.0010. Spot counts for each image are D1: n = 111, D2: n = 165, D3: n = 284, D4: n = 444, D5: n = 192, D6: n = 70, D7: n = 70, D8: n = 69, D9: n = 115, D10: n = 177, N1: n = 132, N2: n = 308, N3: n = 320, N4: n = 88, N5: n = 116, N6: n = 191, N7: n = 209, N8: n = 109, N9: n = 86, N10: n = 150.
