Figure 2
From: Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi
Analysis of STAT3 activation using homoFluoppi. (a) Fluorescent punctate intensity of cells expressing the indicated homoFluoppi constructs without OSM. Each data point represents the mean of eight replicates, and the error bars represent the standard deviation from the mean. Statistical significance was examined in the Student’s t-test with Bonferroni correction (*p < 0.05 versus WT; N.S., not significant). (b) Dose (OSM)-response (fluorescent punctate intensity per cell) curve for cells expressing indicated STAT3 homoFluoppi constructs. OSM treatment for 1 h. Fluorescent punctate intensity per cell was obtained by dividing the total mAG1 (green) fluorescent punctate intensity by the total number of cell (nuclei), which had fluorescence in each field of view. Each point represents the mean of four replicates, and the error bars represent the standard deviation from the mean. (c) Fluorescent images of cells expressing the indicated homoFluppi construct with (top) or without (bottom) OSM. Images of mAG1 (green) and Hoechst33342-stained nuclei (blue) were merged. Scale bars, 25 μm. (d) Western blot analysis of cells transiently expressing the indicated STAT3 homoFluoppi constructs. OSM treatment for 30 min. Full-length blots are presented in Supplementary Figures 5. (e) Dose (JAK inhibitor 1)-response (fluorescent punctate intensity per cell with or without 3 ng/mL OSM) curve for cells stably expressing PB1-mAG1-STAT3. JAK inhibitor 1 was added 30 min before OSM stimulation. OSM treatment for 1 h. Each point represents the mean of four replicates, and the error bars represent the standard deviation from the mean. (f) Fluorescent images of cells stably expressing PB1-mAG1-STAT3 with (middle) or without (left) OSM. JAK inhibitor 1 (right) treatment was 30 min before OSM stimulation. Images of mAG1 (green) and Hoechst33342-stained nuclei (blue) were merged. Scale bars, 25 μm. (g) Western blot analysis of HEK 293 cells (left) or cells stably expressing PB1-mAG1-STAT3 (right). Cells were treated with the indicated concentration of JAK inhibitor 1 for 30 min, followed by treatment with or without 3 ng/mL OSM for 30 min. Full-length blots are presented in Supplementary Figure 5.
