Fig 2: CircKIF4A influences NKTL via the circKIF4A-miR-1231-PDK1/BCL11A axis. (A,B) Predicted miR-1231 binding sites in the 3’ UTRs of BCL11A and PDK1. (C,D) Cells were transfected and subjected to the luciferase assay. (E) The expression of BCL11A and PDK1 was suppressed after transfecting HANK-1 and KHYG-1 cells with miR-1231. (F) Cells were transfected and subjected to Ago2-based RIP.

Fig 3: DHT significantly reverses the antitumor effect in BCL11A-knockdown triple-negative breast cancer cells. (A) Western blot analysis of BCL11A expression after knockdown with si-BCL11A in MDA-MB-231 and Hs578T cells were conducted. Representative blots are presented. (B) Proliferative ability of MDA-MB-231 and Hs578T cells was detected by the colony formation analysis after infection with si-BCL11A and DHT. Colony numbers were counted and were compared among the mock group, si-BCL11A group and si-BCL11A + DHT group. (C) Wound healing assay was applied to measure the migratory ability of MDA-MB-231 and Hs578T cells after infection with si-BCL11A and DHT. (D) Transwell analysis was conducted to measure the invasive ability of MDA-MB-231 and Hs578T cells after infection with si-BCL11A and DHT. Cells were added to transwell inserts and allowed to migrate for 24 h. Representative images are presented. Magnification, ×1100. *P<0.05. BCL11A, B-cell lymphoma/leukemia 11A; si, small interfering; DHT, dihydrotestosterone.

Fig 4: BCL11A-knockdown reduces triple-negative breast cancer cell proliferation. (A) Western blot analysis of BCL11A expression after knockdown with LV-sh-BCL11A in MDA-MB-231 and Hs578T cells were conducted. Representative blots are presented. (B) Flow cytometry was used to detected the percentage of G0/G1, S and G2 phase of MDA-MB-231 and Hs578T cells after LV-sh-BCL11A infection. Green, red and black bars represent cells in the S, G2 and G0/G1 phase, respectively. (C) Proliferative ability of MDA-MB-231 and Hs578T cells was detected by the colony formation analysis after infection with LV-sh-BCL11A. Colony numbers were counted and were compared among the mock group, sh-NC group and sh-BCL11A group. ***P<0.001. BCL11A, B-cell lymphoma/leukemia 11A; sh, short hairpin; NC, negative control; LV, lentivirus.

Fig 5: BCL11A and SOX2 occupy independent and common loci in the genome of LUSC cells. a and b Western blot showing SOX2 and BCL11A expression in SOX2-KD in LK2 (a) and H520 (b) cells transfected with control (scramble), shRNA1 or shRNA2 vectors. c and d BCL11A expression in SOX2-KD LK2 (c) and H520 (d) cells. e Western blot showing BCL11A rescue in SOX2-KD cells. Doxycycline (Dox) inducible BCL11A overexpression vector was transfected into control and SOX2 shRNA1 LK2 cells and Dox treatment was performed for 48 h. f Graph depicting 3D matrigel assay in control, SOX2-KD and BCL11A rescue cells indicating a partial rescue in SOX2-KD, BCL11A overexpressing cells. g Graph indicating partial rescue of tumour size from BCL11A overexpressing SOX2-KD cells injected subcutaneously. The whiskers indicate the range of the data and the line represents the median. h Images of actual tumours measured. Four mice per cell line were monitored for 15 days after which tumours were removed and measured. Data presented as mean ± s.d. (n = 4). One-way ANOVA test performed, *p < 0.05, **p < 0.005, and ***p < 0.001. i LK2 cell line either transfected with control or shRNA1 vectors were used for BCL11A and SOX2 ChIP-Seq. Heatmaps showing BCL11A only, SOX2 only or common peaks in BCL11A or SOX2 IP in control and BCL11A-KD cells. j Venn diagram indicating the overlap of BCL11A and SOX2 target genes in LK2 cells. BCL11A target genes were derived by BCL11A IP in LK2 control cells. SOX2 target genes were derived from SOX2 IP in LK2 control cells. Image below show top five biological GO terms from GO analysis performed using DAVID. k and l IGV genome browser views for SETD8, SKIL, TBX2 and BCL11A