Skip to main content
. 2022 Aug 24;25(9):105011. doi: 10.1016/j.isci.2022.105011

Figure 1.

Figure 1

The CTCF DBD undergoes self-interaction in vitro and in cells with optoDroplet

(A) Schematic illustration of the optoDroplet reporter (left) and blue light-induced target protein domain clustering in live cells (right).

(B) Images of HEK293T cells expressing mCherry-Cry2, FUSN, or the DBD of CTCF fused to mCherry-Cry2 (opto). Representative images of light-activated cells are shown. Fluorescent proteins expressed at similar levels were activated under identical conditions. The percentages of cells forming protein clusters are shown in the bar graphs. Y indicates observed clusters; N indicates that no clusters were observed. Fluorescent signals for the protein expression level used in the optoDroplet assay are shown at the bottom right. Data are represented as the mean ± SD. At least n = 52 cells were used for the calculation. Scale bars, 5 μm.

(C) A CTCF DBD-opto cluster fusion event is shown, with a higher-resolution image below. Scale bars, 5 μm.

(D) Representative images of an FRAP experiment with CTCF DBD-opto in HEK293T cells. Red boxes indicate bleached clusters (left). Quantitative analyses of FRAP data of a CTCF DBD-opto cluster (right). A bleaching event occurred at t = 0 s. Data are represented as the mean ± SD (n = 5). Scale bars, 5 μm, Apparent D: apparent diffusion coefficient; t1/2: half-time of recovery.

(E) Representative images of CTCF DBD-opto in HEK293T cells treated with 3% 1,6-hexanediol for 90 and 180 s (left). Box plot illustration of the fold change in the number of CTCF DBD-opto clusters under 1,6-hexanediol treatment (right). n = 20 in the control and n = 20 in the 1,6-hexanediol treatment group were used for calculation. p-values were calculated using the unpaired two-tailed Student’s t-test (ns not significant, ∗<0.05, ∗∗∗∗<0.0001). Scale bars, 5 μm.

(F) Schematic illustration of the recombinant eGFP and CTCF DBD-eGFP used here (left). Turbidity analyses of the CTCF DBD and eGFP in buffer (20-mM Tris-HCl, 150 mM NaCl) at a concentration of 10 μM at the room temperature (right).

(G) Representative images of droplet formation in the presence of different protein concentrations: CTCF DBD-eGFP or EGFP (bottom). Scale bars, 24 μm.

(H) Representative images of droplet fusion events and photobleaching recovery at the indicated time points. Scale bars, 5 μm (Fusion)/2.5 μm (FRAP).

(I) Representative images of CTCF DBD-eGFP droplet formation in the presence of different concentrations of NaCl. Scale bars, 24 μm.

(J) Representative images of CTCF DBD droplet formation after treatment with 1,6-hexanediol (left) and absorbance analyses at 395 nm (A395) of CTCF DBD proteins in phase separation buffer (right). p-values were calculated using an unpaired two-tailed Student’s t-test (∗∗∗<0.001). Scale bars, 24 μm. Data are represented as the mean ± SD.