Fig 2: ONECUT2 overexpression enhances malignant growth, migration, invasion and adhesion of A549 cells in vitro. (A)Western Blot of whole cell lysates from A549-ONECUT2 and A549-E.V. cells using antibodies against HA and GAPDH. The full-length blot is presented in Supplementary Fig. 5. (B) Light microscopic visualization of A549-ONECUT2 and A549-E.V. cells; scale bar: 50 µm. (C) Two-dimensional cell proliferation assay of A549-ONECUT2 and A549-E.V. cells. Proliferation is measured as absorbance at 490 nm (OD490). Error bars indicate mean ± SD. (D) Soft agar colony formation assay of A549-ONECUT2 and A549-E.V. cells. Images shown are representatives of three independent experiments. (E) Quantification of colonies in soft agar colony formation assay. Error bars indicate mean ± SD (****p < 0.0001, Student’s t-test). (F) In vitro two chamber transwell assay of A549-ONECUT2 and A549-E.V. cells. Images shown are representative of three independent experiments. (G) Quantification of invading cells in the transwell assay. Error bars indicate mean ± SD (****p < 0.0001, Student’s t-test).

Fig 3: ONECUT2 is aberrantly expressed in lung cancer. (A) ONECUT2 expression in various lung cancer subtypes from dataset GSE30219. Data are presented as a Tukey box plot. SQCC: squamous cell carcinoma. (B) Scatter plot of ASCL1 and ONECUT2 expression of SCLC samples from GSE30219. (C) Scatter plot of ASCL1 and ONECUT2 expression of LCNEC samples from GSE30219. (D) Dot plot of Onecut2 and Ascl1 expression in wide type (WT) and Rb1/Trp53 double knockout (Rb1/Trp53 DKO) mice from dataset GSE18534. Error bars indicate mean ± SD. (E) Dot plot of ONECUT2 expression of 58 ADC samples and paired normal tissues from TCGA (****p < 0.0001, paired t-test). (F) Scatter plot of ASCL1 and ONECUT2 expression of ADC samples from GSE30219. (G) Overall survival analysis of ADC patients from TCGA-LUAD stratified by the upper quantile expression level of ONECUT2. (H,I) Overall survival analysis of RAS-driven (H) ADC patients or patients with oncogenic alterations in RTK/RAS/BRAF pathway (I) in the presence of TP53 loss of function alterations from TCGA-LUAD stratified by upper quantile expression level of ONECUT2.

Fig 4: ONECUT2 promotes the xenograft tumorigenesis and bone metastasis of A549 cells in vivo. (A) Schematic view of ONECUT2 and its ?DBD mutant. Cut domain and Homeobox domain along with their position are shown. (B) Western Blot of whole cell lysates from A549-ONECUT2, A549-E.V. and A549-ONECUT2(?DBD) cells using antibodies against HA and a-tubulin. The full-length blot is presented in Supplementary Fig. 5. (C) Quantification of xenograft tumor volume. Error bars indicate mean ± SD (****p < 0.0001, t-test). (D) Image of isolated tumor masses. (E) Scatter dot plot of tumor weight at endpoint. Mean of each group are shown (****p < 0.0001, t-test). (F) Quantification of bioluminescence imaging (BLI) of bone metastasis burden (*p < 0.01, ns: not significant, t-test). (G) In vivo analysis of bone metastasis. Shown are representative mice at 4 weeks after cancer cell implantation; BLI, X-ray, micro-CT and hematoxylin and eosin (H/E) staining of animal hind limbs 6 weeks after cancer cell implantation. Areas circled by dotted lines denote tumor regions, and arrows indicate the regions of bone damage. (H) Overall survival curves of mice injected with different cell lines (*p < 0.01, **p < 0.001, log rank test).

Fig 5: ONECUT2 binds and modulates the activity of bivalent chromatin. (A) Homer motif analysis of high confidence ONECUT2 peaks. (B) GREAT analysis of high confidence ONECUT2 peaks shows distribution of gene-region assignments around TSS. (C) Percentage plot of Roadmap A549 cell chromatin states around activated and repressed ONECUT2 target gene TSS, as well as summits of activating and repressive peaks. Chromatin state around the TSSs of all protein-coding genes and all ONECUT2 target genes are shown as control. (D,E) Plot of average H3K4me3 (D) and H3K27me3 (E) log2 fold change by ChIP-Seq (A549-ONECUT2 versus ?DBD) around activating and repressive ONECUT2 peaks. (F,G) Plot of average H3K4me3 (F) and H3K27me3 (G) log2 fold change by ChIP-Seq (A549-ONECUT2 versus ?DBD) around TSS of genes associated with activating and repressive ONECUT2 peaks. (K) Representative illustration of H3K4me3 and H3K27me3 signals around four selected loci alongside ONECUT2 binding signals in A549-ONECUT2 cells.