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. 2023 Jan 24;14:378. doi: 10.1038/s41467-023-36063-5

Fig. 7. Depletion of the BRD4-NUT/p300 interaction results in EMT signaling suppression.

Fig. 7

a Relative mRNA expression of Snail and EMT markers following transfection of BRD4-NUT-WT or BRD4-NUT-ΔF1c. Bars represent mean +/− SEM (n = minimum of three independent experiments for each condition). Two-tailed unpaired Student’s t-test without adjustment for multiple comparisons. b Western blot analysis of the expression levels of ALX1, Snail, the epithelial cell marker E-cadherin, and the mesenchymal cell markers vimentin and N-cadherin in HEK293T cell overexpressing BRD4-NUT-WT or BRD4-NUT-ΔF1c. Bars represent mean +/− SEM (n = minimum of three independent experiments for each condition). Two-tailed unpaired Student’s t-test without adjustment for multiple comparisons. c Wound healing assay showing cell migration ability of HEk293T cells transfected with empty vector, BRD4-NUT-WT or BRD4-NUT-ΔF1c for 24 h. Migration capabilities were recorded (left) and quantitatively analyzed (right). Scalar bar: 100 μm. The data represent mean +/− SEM (n =  minimum of three independent experiments for each condition). Two-tailed unpaired Student’s t-test without adjustment for multiple comparisons. d qPCR analysis of mRNA transcript levels of MYC, ALX1, NUTM1 in NC 14169 cells transfected with specific siRNA targeting Myc, Alx1, Nutm1 or control (Dharmacon) for 24 h. Bars represent mean +/− SEM (n = minimum of three independent experiments for each condition). Two-tailed unpaired Student’s t-test without adjustment for multiple comparisons. e Western blotting analysis of protein expression levels of Involucrin and GAPDH in NC 14169 cells transfected with indicated siRNA for 72 h. The data represent mean +/− SEM (n = minimum of three independent experiments for each condition). Two-tailed unpaired Student’s t-test without adjustment for multiple comparisons. f A model diagram illustrating a feed-forward loop mechanism propagating BRD4-NUT/p300 chromatin condensation in activating transcription of anti-differentiation and EMT genes including ALX1 in NC cells. Deletion of the TAD1 and TAD2 domains lowers p300 occupancy at ALX1 gene locus and results in downregulation its gene transcription.