Figure. 3. FOSL1-assoicated SEs control CD276 transcription in HNSCC.

(A) MED1 and BRD4 enrichments show that SEs is associated with CD276 in HNSCC. Arrow indicates the AP1 binding site within SEs.

(B) Disrupting SEs by JQ1 and iBET-151 reduces CD276 mRNA expression in HNSCC cells. ***p<0.001 by one-way ANOVA.

(C) Disrupting SEs by JQ1 and iBET-151 reduces CD276 expression in HNSCC cells.

(D) RT-qPCR showing knockdown of FOSL1 by siRNA in HNSCC cells. ***p<0.001 by one-way ANOVA.

(E) Knockdown of FOSL1 inhibited CD276 mRNA expression in HNSCC cells. ***p<0.001 by one-way ANOVA.

(F) Knockdown of FOSL1 inhibited CD276 expression in HNSCC cells by Western blot.

(G) Knockdown of FOSL1 reduced FOSL1 occupancies on SEs in CD276. **p<0.01 by Student’s t test.

(H) Knockdown of FOSL1 reduced MED1 occupancies on SEs in CD276. ***p<0.001 by Student’s t test.

(I) Knockdown of FOSL1 reduced BRD4 occupancies on SEs in CD276. **p<0.01 by Student’s t test.

(J) Over-expression of CD276 induced the expression of c-Jun and FOSL1 in SCC1 and SCC23 cells.

(K) Knockdown of CD276 inhibited the expression of c-Jun and FOSL1 in HN6 cells. Scr, scramble shRNA; C1, CD276 shRNA vector 1, C2, CD276 shRNA vector 2.

(L,M) Representative image and quantification of invasive HN6 and SCC1 cells transduced with scramble shRNA (Scr.); CD276 shRNA1(C1); CD276 shRNA2 (C2). ***p<0.001 by one-way ANOVA. Scale bar, 200μm

(N) The knockdown of CD276 in HN6 cells inhibited tumor growth in nude mice. C2, HN6 cells transduced with CD276 shRNA2; Scr, HN6 cells transduced with scramble shRNA. *p < 0.05 by Student’s t test, n=8. Scale bar, 5mm.

(O) The knockdown of CD276 in HN6 cells inhibited lymph node metastasis. The percentages of lymph node with metastatic tumor cells were analyzed by Fisher’s exact test. ***p < 0.001. Met−, without lymph node metastasis; Met+, with lymph node metastasis. Scale bar, 200μm