Journal: Cell reports

Article Title: ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation

doi: 10.1016/j.celrep.2020.108366

Figure Lengend Snippet: (A) Western blot analysis as indicated in 12Z cells, representative of 2 independent experiments. (B) Invasion of 12Z following indicated treatments. Representative images and total invaded cell numbers are shown (scale bar, 500 μm). Means ± SDs, n = 3. Unpaired, 2-tailed t test. (C) Survival of mice based on time until vaginal bleeding. LtfCre 0/+ ; (Gt)R26Pik3ca * H1047R ; Arid1a fl/fl (n = 16) median (μ 1/2 ) 107 days. LtfCre 0/+ ; (Gt) R26Pik3ca * H1047R ; Arid1a fl/fl ; Ep300 fl/fl (n = 12) median 143 days (p = 0.0018, Mantel-Cox test). LtfCre 0/+ ; Ep300 fl/fl mice were aged to 187 days, and no phenotypes were observed (n = 6). (D) Histology and IHC using indicated antibodies (n ≥ 2 mice) in endometrium (scale bar, 200 μm). KRT8 was a positive control for endometrial epithelium. The arrowheads indicate epithelia. (E) Quantification of H3K27ac and H3K18ac IHC, ratio of H-scores of epithelia to stroma. Means ± SDs, n = 4–8 mice, unpaired, 2-tailed t test. (F) Western blot of H3K27ac following A-485 treatment of 12Z for 24 h and densitometry of H3K27ac relative to H3, normalized to control (vehicle). Means ± SDs, n = 3–5 independent replicates per condition. Unpaired, 2-tailed t tests were performed in comparison to the vehicle treatment condition. Irrelevant lanes were removed from the image; see . (G) Viability assay for cells treated with A-485, normalized cell counts relative to vehicle control. Raw data are presented in . Half-maximal inhibitory concentration (IC 50 ) values were not significantly different between 12Z untreated and control shRNA, or between control shRNA and shARID1A (unpaired, 2-tailed t test). Means ± SDs, n = 4. (H) Invasion of 12Z following indicated cell treatments. Representative images and total invaded cell numbers are shown (scale bar, 500 μm). Means ± SDs, n = 4. Unpaired, 2-tailed t tests performed in comparison to siARID1A + vehicle. (I) Caspase 3/7 activity of indicated cell treatments. Means ± SDs, n = 3. Unpaired, 2-tailed t test. (J) Ratio of dead to live cells after 16 h in Matrigel. Means ± SDs, n = 6. Unpaired, 2-tailed t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Sections were incubated with antibodies at the following dilutions: 1:200 ARID1A (D2A8U) (12354, Cell Signaling); 1:1000 P300 (86377, Cell Signaling); 1:400 Phospho-S6 (4585, Cell Signaling); 1:100 KRT8 (TROMA1, DHSB); 1:200 Cleaved Caspase-3 (9579, Cell Signaling); 1:400 Ki67 (12202, Cell Signaling); 1:200 H3K27ac (39133, Active Motif); 1:200 H3K18ac (ab1191, Abcam); 1:1000 PAI-1 (SERPINE1) (ab66705, Abcam).

Techniques: Western Blot, Positive Control, Viability Assay, Concentration Assay, shRNA, Activity Assay