Figure 3.

MTA3 Inhibits SOX2OT Transcription Depending on GATA3

(A) QRT-PCR of SOX2OT in EC9706 cells with MTA3 depletion or MTA3 overexpression and in HKESC-1 cells with MTA3 overexpression.

(B–D) Western blot of SOX2 and qRT-PCR of SOX2OT in tumors derived from EC9706 (B) and EC109 (C) cells with MTA3 depletion, or EC9706 cells and HKESC-1 cells with MTA3 overexpression (D).

(E) SOX2OT Gaussia luciferase reporter activity in EC9706 cells with MTA3 depletion or overexpression.

(F) Schematic structure of the SOX2OT promoter and positions of ChIP primers.

(G) Proximity Ligation Assay (PLA) detection of MTA3-GATA3 interaction. EC9706 cells transfected with the indicated shRNAs were subjected to PLA using antibodies against MTA3 or GATA3. Scale bars: 10 μm.

(H and I) ChIP assay using antibodies against MTA3 or IgG. Semi-quantitative PCR (H) and qPCR (I) to detect the enriched DNA fragments in the SOX2OT promoter region.

(J) Western blot of GATA3 in MTA3 overexpressed EC9706 cells transfected with shGATA3-expressing plasmid. β-Actin is shown as a loading control.

(K) SOX2OT luciferase reporter activity in EC9706 cells transfected with the MTA3 or shGATA3 plasmids.

(L) ChIP assay was performed in EC9706 cells with GATA3 depletion using antibodies against MTA3 or IgG, and qPCR was used to detect the enriched DNA fragments in the SOX2OT promoter region.

(M) Schematic structure of deletion-mutation reporters of the SOX2OT promoter.

(N and O) The SOX2OT promoter-reporter and mutations ΔGATA3 (N) or in GATA3-binding site 1 (ΔGATA3 #1), GATA3-binding site 2 (ΔGATA3 #2), GATA3-binding site 3 (ΔGATA3 #3) (O). The relative SOX2OT Gaussia luciferase reporter activities 72 h after transfection.

Data are shown as the means of three independent experiments or representative data. Error bars indicate SEM, n.s., not statistically significant; *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test or a one-way ANOVA with post hoc intergroup comparisons, where appropriate. See also Figures S6 and S7, and Table S3.