Fig 1: Contribution of the TLK2-DYNLL1 axis to the inter-transition of non-CSCs to CSCs.a Flow cytometry analysis of ALDH-positive cell rates in MDA-MB-231 cells with or without TLK2 or DYNLL1 knockdown. Means and standard deviation from biological triplicates (n = 3) are shown. (p = <0.0001; <0.0001; <0.0001; <0.0001) b Mammosphere formation assay of MDA-MB-231 cells with or without TLK2 or DYNLL1 knockdown. Scale bar, 50 μm. Means and standard deviation from biological triplicates (n = 3) are shown. (Diameter: p = <0.0001; <0.0001; <0.0001; <0.0001; Number: p = <0.0001; <0.0001; <0.0001; <0.0001) c Flow cytometry analysis of ALDH-positive cell rates in TLK2 knockdown MDA-MB-231 cells with DYNLL1 S14D or S14A mutant rescue. Means and standard deviation from biological triplicates (n = 3) are shown. (p = <0.0001; 0.1406; <0.0001) d Mammosphere formation assay in TLK2 knockdown MDA-MB-231 cells with S14D or S14A DYNLL1 mutant rescue. Scale bar, 50 μm. Means and standard deviation from biological triplicates (n = 3) are shown. (Diameter: p = <0.0001; 0.0505; <0.0001; Number: p = <0.0001; 0.0702; <0.0001) e Limiting dilution assay (LDA) in NOD/SCID mice comparing tumor formation in TLK2 knockdown versus control cells. Resected tumor xenografts were subjected to mammosphere formation assays to evaluate mammosphere-forming capacity. Means and standard deviation from biological triplicates (n = 3) are shown. (p = <0.0001) f ALDH-low (ALDHlo) MDA-MB-231 cells were sorted, re-seeded, and monitored for ALDH-positive rates over 6 days. Means and standard deviation from biological triplicates (n = 3) are shown. (p = 0.0003; 0.0011; <0.0001; 0.0004; 0.0010) g Co-culture of mNeonGreen-labeled control MDA-MB-231 cells with mRuby3-labeled TLK2 knockdown cells under 2D normal or 3D mammosphere conditions, analyzed by flow cytometry for proportional changes. Error bars represent mean ± SD (a, b, c, d, f). Statistical significance was determined by one-way ANOVA with Bonferroni correction (a, b, c, d); two-tailed unpaired Student’s t test (e, f); ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.
Fig 2: The effects of TLK2 inhibition on the formation of CTCF-cohesin hubs in proximity to the stemness-associated gene KLF4.a Venn diagram illustrating candidate stemness genes regulated by the TLK2-DYNLL1 axis. b CTCF ChIA-PET data from H1-hESC (4DN Data Portal) indicating interaction between the KLF4 promoter and an enhancer located within the LINC01509 gene. Red circle presents the potential interaction. c Analysis of RNA expression, chromatin accessibility, and binding of CTCF, RAD21, DYNLL1, and H3K27ac at the KLF4 locus in TLK2 knockdown versus control cells. The merged peaks are shown from two biological replicates. d, e qPCR and Western blot showing KLF4 expression in TLK2 and DYNLL1 knockdown cells. Means and standard deviation from biological triplicates (n = 3) are shown. (p = <0.0001; <0.0001; <0.0001; <0.0001) f Chromosome conformation capture (3C-qPCR) assays measuring interaction frequency between the KLF4 promoter and its enhancer. Means and standard deviation from biological triplicates (n = 3) are shown. (p = 0.0003; 0.0019; 0.0030; 0.0324) Error bars represent mean ± SD (d, f). Statistical significance assessed by one-way ANOVA with Bonferroni correction (d, f); ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.
Fig 3: Model of TLK2 suppression in disrupting CTCF-cohesin hub formation to target cancer stemness plasticity.Upper left, Schematic summarizing the identification of TLK2 as a regulator of CTCF-cohesin hub formation through CTCF-cohesin hub reporter and CRISPR screening. Upper right, Schematic illustrating the role of the TLK2-DYNLL1 axis in regulating CTCF-cohesin hub formation in proximity to the KLF4 stemness gene. Lower, Schematic depicting the suppression of CTCF-cohesin hub formation by targeting TLK2, inhibiting cancer stemness plasticity.
Supplier Page from Novoprotein Scientific for Recombinant Human DYNLL1 (N-6His)