Fig 1: Disrupting SEs suppresses the tumorigenic potential and metastasis of CSCs from human HNSCCs.a Representative FACS plots of CD44highALDHhigh cells in control and JQ1-treated SCC1 cells. Values are mean ± SD for triplicate experiments Statistical analysis was performed using two-tailed unpaired Student’s t-test. b Representative images of tumorsphere formation assays for isolated CD44highALDHhigh cells upon JQ1 treatment. Scale bar, 200 µm. c Quantification of tumorspheres from isolated CD44highALDHhigh cells upon JQ1 treatment. Values are mean ± SD for triplicate experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-test. d Representative FACS plots of EpCAM+CD44highALDHhigh CSCs from human PDXs of HNSCC. e Representative images of tumorsphere formation assays for isolated EpCAM+CD44highALDHhigh CSCs upon JQ1 treatment. f Quantification of tumorspheres from EpCAM+CD44highALDHhigh CSCs upon JQ1 treatment. Values are mean ± SD for triplicate experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-test. g Representative FACS plots and quantification of tumorspheres of CD44highALDHhigh CSC-like cells in control and JQ1-treated SCC1-cis cells. Values are mean ± SD for triplicate experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-test. h JQ1 inhibited orthotopic tumor growth of human CD44highALDHhigh CSC-like cells from SCC1-cis cells (n = 8 per group). Statistical analysis was performed using two-tailed unpaired Student’s t-test. Scale bar, 1 mm. i Immunostaining of lymph node metastasis with anti-pan cytokeratin antibodies (n = 8 per group; 25 lymph nodes were retrieved from Control group, and 24 lymph nodes were retrieved from JQ1 treatment group). Scale bar, 50 µm. j JQ1 inhibited lymph node metastasis of human CD44highALDHhigh CSC-like cells from SCC1-cis cells (n = 8 per group; 25 lymph nodes were retrieved from Control group, and 24 lymph nodes were retrieved from JQ1 treatment group). Statistical analysis was performed using two-tailed Fisher’s exact test. k JQ1 inhibited orthotopic tumor growth of EpCAM+CD44highALDHhigh CSCs from human PDXs of HNSCC (n = 9 for Control group; n = 8 for JQ1 treatment group). Statistical analysis was performed using two-tailed Student’s t-test. Scale bar, 1 mm. l Immunostaining of metastatic tumor cells in cervical lymph nodes using anti-pan-keratin (n = 9 for Control group, 37 lymph nodes were retrieved from this group; n = 8 for JQ1 treatment group, 36 lymph nodes were retrieved from this group). m JQ1 inhibited lymph node metastasis of human EpCAM+CD44highALDHhigh CSCs from human HNSCC samples (n = 9 for Control group, 37 lymph nodes were retrieved from this group; n = 8 for JQ1 treatment group, 36 lymph nodes were retrieved from this group). Statistical analysis was performed using two-tailed Fisher’s exact test. Scale bar, 50 µm. The experiments in h–m were repeated once with similar results.