Fig 2: SOX2 enables JAK–STAT activation in a positive feedback fashion.a–d, SOX2 ChIP–qPCR of JAK1 (a,c) and STAT1 (b,d) genomic loci in LNCaP/AR cells transduced with annotated CRISPR guide RNAs or overexpressing constructs. e, Relative cell numbers of LNCaP/AR cells transduced with annotated constructs and treated with Enz or vehicle, normalized to the vehicle group; Enz, 10 μM Enz; vehicle, DMSO treatment with equal volume as Enz. Cells were treated for 6 d, and cell numbers were measured by cell proliferation assay. f, Relative cell number fold change of LNCaP/AR cells transduced with annotated constructs. Data are normalized to the SOX2-OE + sgNT group; Enz, 10 μM Enz treatment for 8 d. Cell numbers were measured by a CellTiter-Glo assay. P values were calculated by one-way ANOVA with a Bonferroni multiple-comparison test. g, Relative expression of canonical lineage marker genes in LNCaP/AR SOX2-OE cells transduced with annotated constructs. h, Relative expression of canonical lineage marker genes in LNCaP/AR cells transduced with JAK1 or STAT1 cDNA constructs. SOX2 expression is highlighted in red. i, Relative expression of SOX2 in LNCaP/AR cells transduced with annotated guide RNAs. j, Relative expression of SOX2 in LNCaP/AR cells treated with 5 µM Filg or 5 µM ruxolitinib (Rux) or DMSO for 8 d. P values in i and j were calculated by one-way ANOVA with a Bonferroni multiple-comparison test. k, Relative gene expression levels of canonical JAK–STAT signaling and lineage marker genes in the inducible shTP53/RB1 LNCaP/AR cells treated with Dox for various lengths of time. Data are normalized to 0 h. Unless otherwise noted, n = 3 independent treated cell cultures, and data represent mean ± s.e.m. P values were calculated by two-way ANOVA with a Bonferroni multiple-comparison test.Source data

Fig 3: A CTCF motif insertion between the Sox2 gene and the SCR disrupts the SCR–Sox2 gene interaction, but not SCR-mediated enhancer activity. (A) The region surrounding the Sox2 gene is displayed on the UCSC genome browser (mm10). The Sox2 control region (SCR) is shown along with the orientation of the CTCF motifs within CTCF-bound regions. The vertical blue line represents the location into which the sequences shown above were integrated. CTCF and RAD21 ChIP-seq conducted in ESCs is shown below. In B and C, 4C data are shown for no CTCF integration (black), CTCF sites integrated in an orientation convergent with the SCR site (green), and CTCF sites integrated in an orientation convergent with the Sox2 site (brown). The vertical blue line represents the location into which the sequences were integrated, and the dashed line marks the location of the 4C bait at the SCR (B) or the integration site (C). Gray boxes indicate the bait-interacting regions where significant differences were identified surrounding the Sox2 gene (blue arrow), SCR (black box), or integration site (blue line). Compared with control cells lacking CTCF at the insertion site, a significant decrease in relative interaction frequency of the 4C bait region at the SCR with the Sox2 gene was observed in the SCR-convergent CTCF insertion (P = 0.002), but not the Sox2-convergent CTCF insertion (P = 0.1). Compared with control cells, a significant increase in relative interaction frequency of the 4C bait region at the insertion with the Sox2 gene site was observed in the SCR-convergent CTCF insertion (P = 0.02) and the Sox2-convergent CTCF insertion (P = 0.005). A significant increase in relative interaction frequency of the 4C bait region at the insertion with the SCR was observed in the SCR-convergent CTCF insertion (P = 0.02) but not the Sox2-convergent CTCF insertion (P = 0.6). (D) Allele-specific primers detect Sox2 musculus (129) or castaneus (CAST) mRNA by RT-qPCR from F1 ESC clones from the genotype indicated. Expression levels from either allele are shown relative to the total transcript levels. Error bars represent SD. n = 2–3. (ns) Not significant (P > 0.05). (E) Maximal projections of representative micrographs from smFISH experiments performed on ESCs with only the control sequence, or with the control sequence and CTCF sites convergent with the SCR, introduced at the insertion site. Sox2 mRNA molecules are labeled in red, with DAPI staining in blue. Scale bar, 10 μm. (F) Violin plot comparing distributions of Sox2 mRNA molecules per cell in ESCs with only the control sequence (black; n = 819 cells from two replicates), or the control sequence plus CTCF sites convergent with the SCR (green; n = 1289 cells from two replicates), at the insertion site. (G) Proportions of cells with zero, one, or more than one active transcription sites (TSs) for ESCs with only the control sequence (black), or the control sequence plus CTCF sites convergent with the SCR (green), at the insertion site.

Fig 4: Gene expression patterns in A549 cell spheroid.Notes: (A) Quantitative real-time RT-PCR of transcript levels for EMT markers (CDH1, CDH2, SNAI1, and ZEB1), cell cycle markers (MKI67, GADD45A, and CDKN1A), and pluripotent markers (OCT4, SOX2, KLF4, C-MYC, and NANOG). Data shown are representative of two independent experiments, and the values represent the mean ± SD of triplicate samples. The expression of each mRNA was normalized to that of GAPDH mRNA in the same sample and is presented as the fold-change over that of 2D culture control cells. Differences in expression levels were evaluated for significance using one-sided t-tests with unequal variance (*P < 0.01, **P < 0.05, ***P < 0.001). (B) The protein expression changes were estimated by Western blot. β-Actin was used as an internal control.Abbreviations: EMT, epithelial-to-mesenchymal transition; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 2D, two dimension; 3D, three dimension.

Fig 5: Androgen deprivation causes direct binding of SOX2 on the H19 promoter upregulating its transcription during NEtD.A H19 RNA expression during different phases of NEtD in the LTL331 system (n = 1). B Relative RNA expression of H19 and PSA in C4-2B cells after DHT (10 ng, 48 h) treatment vs. control (EtOH). C Same as B after ENZA (10 µM, 5 days) treatment vs. DMSO. D Relative RNA expression of H19 in androgen starved (AR−) LNCaP cells placed in phenol-free media with 10% CSS for 40 days. LNCaP cells grown in regular media (AR+) are controls. The right panel demonstrates WB assay for indicated proteins and Actin (control) using cells in the left. E Genomic location of AR binding motifs (ARE) on H19 promoter. F In C4-2B cells vs. control (DMSO) cells, ChIP-qPCR showed AR binding on H19 promoter and a decrease with ENZA (10 µM) treatment. The KLK3 promoter served as a positive control. G Schematic showing luciferase reporter construct with the proximal promoter of H19 (H19-PP) harboring ARE sites (ARE 3–4) (top). Luciferase reporter activity of the H19-PP in DHT (10 nM) stimulated cells vs. ethanol (control) treatment. The right panel is the same, except for ENZA (10 µM) treatment of C4-2B cells. H Relative RNA expression of SOX2 and H19 in C4-2B cells stably expressing sh-SOX2 vs. sh-SCR control (top); and stably expressing SOX2 vs. empty vector control transduced cells (bottom). I Genomic location of SOX2 binding motifs (S-1, S-2) on the H19 promoter. Bar plots demonstrating ChIP-qPCR are showing enrichment of SOX2 on the H19 promoter upon ENZA (10 µM) and DMSO (control) treatment in C4-2B cells (bottom). J Schematic showing luciferase reporter constructs with H19-PP wild-type (S-1 WT) or mutated (S-1 Mut) SOX2 binding site (mutation in blue) (top). Bar plots showing luciferase reporter activity of H19-PP WT-S1 or Mut-S1 in ENZA (10 µM) treated 42DENZR cells (bottom). K Diagram depicting the regulation of H19 during NEtD mediated by androgen deprivation (ADT) and consequently via SOX2. Data are mean ± SD (B, D, F–J), or mean ± SEM (C); n = 3 (B, C, D, H) or n = 4 (F, I, J) biological replicates. p Values were calculated by unpaired two-tailed Student’s t test (B–D, G–I) or two-way ANOVA multiple comparisons test (F, J).