(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