Skip to main content
. Author manuscript; available in PMC: 2019 Apr 3.
Published in final edited form as: Cell Rep. 2019 Mar 26;26(13):3629–3642.e4. doi: 10.1016/j.celrep.2019.02.108

Figure 2. Permeabilization-Fixation Retains Chromatin-Associated GRs.

Figure 2.

(A) GFP-GR 3617 cells were treated with corticosterone, kept on ice (Live), fixed or perm-fixed, and data were acquired by imaging flow cytometry. DAPI staining was used to define the nucleus after fix or perm-fix. Frequencies of GR+ nuclei were quantified using the similarity score algorithm included in the IDEAS analysis software. Images are from cells with the median GFP/DAPI similarity score from each sample. Data are representative of three independent experiments. Scale bar, 10 μm.

(B) GFP-GR 3617 cells were treated with corticosterone, perm-fixed, washed, diluted in SDS gel loading buffer, subjected to SDS-PAGE, and immunoblotted for the indicated proteins. Blots are representative of two independent experiments.

(C and D) 3617 cells expressing the wild-type GR (GFP-GRwt), dimerization-impaired GR (GFP-GRmon), or deficient for GR (KO) were treated with different concentrations of corticosterone, kept on ice (C), or perm-fixed (D), and data were acquired by flow cytometry. Data are representative of two independent experiments.

(E) HEK293T cells were transfected with plasmids encoding the wild-type GR (GFP-GRwt), GR C440G mutant (GRC440G), which has reduced DNA binding, or the GRC440G/mon double mutant. GFP+ cells were sorted, treated with different concentrations of corticosterone, and perm-fixed, and data were acquired by flow cytometry. Live cells were untreated. Data are representative of three independent experiments.

(F) GFP-GR 3617 cells were treated with corticosterone for 15 min and then incubated in steroid-free medium for the indicated times and perm-fixed, and data were acquired by flow cytometry. The GR MFI of cells perm-fixed in the presence of corticosterone was set to 100%, and the MFI of cells kept in steroid-free medium was set to 0%. Data are means ± SEM of three independent experiments.

(G) GFP-GR 3617 cells were treated with corticosterone, incubated in steroid-free medium for 15 min, and perm-fixed, and data were acquired by imaging flow cytometry. Data are representative of two independent experiments. Scale bar, 10 μm.