JCS035865 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Example of automated image analysis. (A) Overlay images of GFP-GR signal (green) and infrared RNA FISH signal (blue) are shown for three cell line 3617 cells treated for 0.5 hour with Dex. The yellow square indicates the region of the nucleus, which contains the MMTV array. This small boxed region is expanded in the images below, so that individual pixels can be distinguished (B-E). Each pixel represents 111 nm in the cell. The displayed pixel intensities have not been smoothed or interpolated, and displayed pixel to pixel variation is identical to that in the automatically measured images. (B) Grey scale images show the fluorescence intensity of the RNA FISH signal for each cell. The automatically defined boundary pixels for the FISH signal region of interest (ROI) are shown in yellow. (C) Grey scale images show the fluorescence intensity of the GFP-GR signal. The boundary pixels from the FISH signal ROI are shown in yellow for reference. The positional information of the FISH ROI is used to find the maximal pixel intensity of GFP-GR fluorescence associated with the array. Two different GFP-GR loading ratios are shown for each cell. As indicated, the loading ratios are calculated based on the intensity of the single maximal pixel intensity or based on the mean intensity of a 3×3 pixel area centered on the maximal pixel. (D) To highlight the differences in relative concentration of GFP-GR localized at the array, the GFP-GR fluorescence intensity is also represented with pseudo color. (E) This same pseudo color scale also compares the relative concentration of GFP-GR associated with the MMTV array in 3D projections of the fluorescence intensity.

• Supplemental Figure S2 -

  Fig. S2. The loading ratio derived from the maximal intensity pixel of GFP-GR associated with the MMTV array correlates with the loading ratio derived from the mean intensity of the immediately surrounding pixels. The plots show the loading ratio based on the single maximal pixel (as described in the Materials and Methods) compared with the loading ratio based on the mean intensity value of a 3×3 pixel square ROI (9 pixels total), which is centered on the maximal pixel. Each point represents the loading ratio values from a single cell. The red line is the best-fit linear relationship for all cells calculated by linear regression. The two types of loading ratios (single pixel max, and 9 pixel surrounding) are correlated for GFP-GR and the Ch-Coregulators in the indicated cell lines following 30 minutes hormone treatment. The R² values show the degree of correlation between the two types of loading ratios.

• Supplemental Figure S3 -

  Fig. S3. Relative expression levels of XFP-fusion proteins. Western blots are shown for the indicated stable-inducible cell lines grown for 18 hours in the presence of Tet or the absence of Tet to induce expression of the XFP-fusion proteins. Specific antibodies were used to detect (A) GR, (B) Brg1 and (C) GRIP. As a loading control, the Ponceau S stained membrane for each western blot is shown. Based on this analysis, GFP-GR is expressed at approximately a quarter of endogenous GR levels in cell line 6643. Mean nuclear fluorescence intensity for each cell population suggests that GFP-GR is expressed at identical levels in all three cell lines (data not shown). Ch-Brg1 is expressed at approximately equal levels relative to the endogenous protein. Cell line 6790 expresses approximately twice as much Ch-GRIP compared with endogenous levels. These data indicate that the XFP-fusion proteins are not massively overexpressed in these stable-inducible cell lines.

• Supplemental Figure S4 -

  Fig. S4. Statistical comparison of GFP-GR array loading ratio and RNA FISH loess lines in the 3617 and the 6643 cell lines. The individual plots (A-F) display each pairwise comparison of loess lines, which are also shown in Fig.5A. The P value indicating the statistical difference between the loess lines is calculated for the individual values of GFP-GR loading ratio by implementing a permutation test, and the P values are plotted (black diamond markers) along with the Loess lines being compared. If P is below the black horizontal line (P<0.05), then the loess lines are significantly different at that GFP-GR loading ratio value.

• Supplemental Figure S5 -

  Fig. S5. Statistical comparison of GFP-GR array loading ratio and RNA FISH loess lines in the 3617 and the 6790 cell lines. The individual plots (A-F) display each pairwise comparison of loess lines, which are also shown in main Fig. 5B. The P value indicating the statistical difference between the loess lines is calculated for the individual values of GFP-GR loading ratio by implementing a permutation test, and the p-values are plotted (black diamond markers) along with the loess lines being compared. If the P value is below the dotted horizontal line (P<0.05), then the Loess lines are significantly different at that GFP-GR loading ratio value.

• Supplemental Figure S6 -

  Fig. S6. Statistical comparison of Ch-coregulator array loading ratio and RNA FISH loess lines in the 6643 and the 6790 cell lines. The individual plots (A,B) display pairwise comparison of loess lines, which are also shown in Fig. 5C,D. The P value indicating the statistical difference between the loess lines is calculated for the individual values of Ch-coregulator loading ratio by implementing a permutation test, and the P values are plotted (black diamond markers) along with the P value being compared. If the P value is below the dotted horizontal line (P<0.05), then the loess lines are significantly different at that Ch-coregulator loading ratio value.

• Supplemental Figure S7 -

  Fig. S7. Statistical comparison of GFP-GR array loading ratio and Ch-coregulator array loading ratio loess lines in the 6643 and the 6790 cell lines. The individual plots (A,B) display each pairwise comparison of loess lines, which are also shown in Fig. 6. The P value indicating the statistical difference between the loess lines is calculated for the individual values of GFP-GR loading ratio by implementing a permutation test, and the P values are plotted (black diamond markers) along with the loess lines being compared. If the P value is below the dotted horizontal line (P<0.05), then the Loess lines are significantly different at that GFP-GR loading ratio value.