Supplementary Figure 3: SMARCA4-Dendra2 is highly stable and undergoes dynamic changes in interaction with chromatin throughout the cell cycle.
From: Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers
(a) Following photoconversion with 405-nm light using a custom-made LED lamp, SMARCA4-Dendra2 irreversibly photoswitches from green to red. (b) Photoswitching is readily detectable using flow cytometry. (c) The persistence of red fluorescence provides a measure of the stability of the protein fusion. Red fluorescence persists with a half-life of 11.8 ± 1.2 h, comparable to the canonical 12-hour half-life of mESCs. This result indicates that fluorescent fusions of SMARCA4 are stable. (d) Live-cell imaging of mitosis. Mitotic cells are readily distinguishable from interphasic cells due to the altered distribution of fluorescence intensity. (e) Fluorescence recovery after photobleaching (FRAP) of an individual mESC colony. (f) FRAP recovery times of SMARCA4-Dendra2 fusions are faster in mitosis, when the complex is excluded from condensed mitotic chromatin (g) Statistical comparison of FRAP recovery times of SMARCA4-Dendra2 fluorescent fusions. Mitotic cells show significantly increased FRAP recovery times (p<0.002, KS test). (h) Custom LED array for bulk photoconversion of cells using 405-nm LEDs (DigiKey 492-1349-ND).
