Fig 2: Inhibin‐α‐expressing cells showed immature property. (A) Representative images of immunofluorescence double staining (anti‐inhibin‐α and anti‐ALDH1A1 antibodies). Scale bars: 20 μm. (B) Representative immunoblotting of ALDH1A1 and inhibin‐α in 2D and 3D cells. β‐actin and CBB staining were used for loading controls. (C) Proportion of ALDHhigh cells according to the ALDEFLUOR assay. Data are shown as mean ±SE from at least three independent experiments (**p < 0.01 by the Steel–Dwass nonparametric multiple comparison test). (D) Representative immunoblotting of inhibin‐α in sorted ALDHhigh and ALDHlow cells. Inhibin‐α: β‐Actin ratios were calculated based on band intensities measured with densitometry. Data are shown as mean ±SE from at least three independent experiments (*p < 0.05 by the Wilcoxon signed‐rank test). (E) The correlation between inhibin‐α, immature stemness‐related genes, and proliferative MKI67 gene in the GSE129617. (F) Representative images of immunohistochemistry of inhibin‐α using OCCC tissues with high (score 3), intermediate (score 2), low (score 1), or null (score 0) signal intensity, which were used for calculating H‐scores. Scale bars: 50 μm. There were five recurrent cases diagnosed pathologically. OCCC tissue sections at the time of first occurrence and recurrence were stained with anti‐inhibin‐α. Scale bars: 200 μm. (p = 0.0517 by the Wilcoxon signed‐rank test)

Fig 3: Chemoresistant cells showed enriched stemness properties and high inhibin‐α expression. (A) Cell viability (CV) in OCCC cell lines that were treated with CBDCA (Dose: 100 μM). Data were shown as mean ±SE from at least three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001 by the Steel–Dwass nonparametric multiple comparison test). (B) Half maximal Inhibitory concentration for CBDCA was assessed for each cell line. (C) Representative dose response curves of RMGV, RMGV‐R, OVTOKO, and OVTOKO‐R that were treated with CBDCA were shown (upper panel). Corresponding CV values were shown (lower panel). Data were shown as mean ±SE from at least three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001 by the Wilcoxon signed‐rank test). (D) ALDEFLUOR assay was performed on RMGV, RMGV‐R, OVTOKO, and OVTOKO‐R. Data were shown as mean ±SE from at least three independent experiments (*p < 0.05, ***p < 0.001 by the Wilcoxon signed‐rank test). (E) Representative images of spheroids in RMGV, RMGV‐R, OVTOKO, and OVTOKO‐R. Spheroids were counted in five random fields per well. The diameters of all counted spheroids were measured. Scale bars: 200 μm. Data were shown as mean ±SE from at least three independent experiments (**p < 0.01, ***p < 0.001 by the Wilcoxon signed‐rank test). (F) Immunoblotting of ALDH1A1 and inhibin‐α. β‐Actin and CBB staining were used for loading controls. (G) Representative images of cell blocks (RMGV and RMGV‐R). They were stained with anti‐inhibin‐α antibody. Scale bars: 50 μm. Data were shown as mean ±SE from at least three independent experiments (***p < 0.001 by the Wilcoxon signed‐rank test)

Fig 4: The correlation between ALDH1 expression, c-Myc expression, and NEAT1 expression in CRC tissues.A Representative IHC staining of ALDH1 or c-Myc and NEAT1 in serial sections are shown. B NEAT1 overexpression was significantly positively correlated with ALDH1 and c-Myc upregulation in human CRC tissues. Spearman correlation analysis was used. C Combination of ALDH1 or c-Myc and NEAT1 had a higher probability of poor prognosis than either factor alone. P is based on the log-rank test.

Fig 5: Genetic ablation of DLX1 inhibits oncogenic properties.a Q-PCR data showing expression of EMT markers in 22RV1-DLX1-KO and control SCR cells. b Immunoblots showing vimentin and E-cadherin using same cells as a. ß-actin was used as loading control. c Same as b except for cleaved PARP, cleaved Caspase-3 and Bcl-xL. d Flow cytometry-based apoptosis assay using 22RV1-DLX1-KO and control cells (top panel) and DLX1-silenced VCaP cells (bottom panel). e Flow cytometry data for cell cycle distribution using same cells as in d. f Q-PCR data for stem cell markers using same cells as d. g Flow cytometry data depicting ABCG2 (top panel) and CD44 (bottom panel) expression in DLX1 overexpressing RWPE1 cells, 22RV1-DLX1-KO, and DLX1-silenced VCaP cells. h Fluorescence intensity of catalyzed ALDH substrate in 22RV1-DLX1-KO and control cells. Marked windows show ALDH1 + percent cell population. i Same as h, except for DLX1 silenced VCaP cells. j Q-PCR data showing expression of target genes in 22RV1-DLX1-KO and control cells(P < 0.0001). k Schema showing the chromosomal location of DLX1 binding motif (DBM1/2) at the ALDLH1A1 (top) and HNF1A (bottom) promoters. l ChIP-qPCR data of DLX1 (top panel) and H3K9Ac (bottom panel) on ALDH1A1 in 22RV1-DLX1-KO and SCR control cells (P < 0.0001). m Same as in l except for the HNF1A promoter. Data shown from three biologically independent samples (n = 3). Data represent mean ± SEM. For panels, a, f, and j Two-way ANOVA, Dunnett’s multiple comparison test; l, m Two-way ANOVA Turkey’s multiple comparison test was applied. Source data are provided as a Source Data file.