Fig 2: Loss of LKB1 clinically associates with the increase of ALKBH5 and decrease of m6A modification on SOX2, SMAD7, and MYC genes.A Representative immunostaining images and quantification of 5mC DNA in lung cancer patients with KW and KM. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. B, C High levels of ALKBH5 DNA methylation and mRNA expression in cases of LKB1 loss compared with that of LKB1 positive expression in KM patients. D–F The correlation of ALKBH5 protein with the global m6A modification (D), m6A levels of SOX2, SMAD7, and MYC genes (E, F) in KM and KW groups. G–I Negative correlations of the m6A enrichment with mRNA expressions of SOX2, SMAD7, and MYC in KM group. J Representative immunostaining images and quantification of SOX2, SMAD7, and MYC in KM group with LKB1 loss or not. K The positive correlations of ALKBH5 with SOX2, SMAD7, and MYC protein expression in KM group. Data as mean ± SD. KRAS mutation (KM). KRAS wild-type (KW). *P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for A, B, and C. Student’s t-test for J.

Fig 3: Oleacein suppresses stemness-related SOX2 protein expression. Western blotting analyses of SOX2 protein levels in human fibroblasts, induced pluripotent stem (iPS) cells, MCF-7, and MCF-7/SOX2 cells cultured in the absence or presence of graded concentrations of oleacein. ß-actin expression was employed as an internal loading control. Images are representative of two independent experiments. Total RNA from human fibroblasts, iPS cells, MCF-7, and MCF-7/SOX2 cells cultured in the absence or presence of graded concentrations of oleacein was characterized in technical triplicates for the relative abundance of SOX2 using RT-PCR analysis. The transcript abundance was calculated using the delta Ct method and presented as fold-change versus basal expression in untreated cells.

Fig 4: SOX2 inhibition induced G1/S cell cycle arrest. a, and b The cell cycle was assayed by flow cytometry. The percentages of cell cycle phases were displayed in bar charts. siSOX2 transfection resulted in a significant increase of the cells proportion in the G0/G1 phases of A673 and RD-ES cells. c SOX2 inhibition amplified the expression of p21 and p27, and repressed cyclin-E, but had no effect on cyclin-D expression in Ewing’s sarcoma cells according to assayed by WB analyses. The data are representative and selected from one of three independent experiments

Fig 5: Generation and identification of engineered hiPSC-derived cortical organoids.a Schematic diagram and bright-field images of hiPSC-derived cortical organoids developed on a chip. Scale bar = 200 µm. b Immunohistochemical image of tissue sections used to detect the apoptosis protein Caspase3, neural progenitor cells (NESTIN and SOX2), differentiated neurons (TUJ1), distinct brain regions (forebrain: FOXG1, PAX6; hindbrain ISL1; developing brain marker PAX2; cortical layer, TBR1 and CTIP2), inhibitory neurons (GABA) and excitatory neurons (vGLUT1) in cortical organoids at 30 and 40 days of differentiation by immunohistochemical analysis. Scale bars = 100 µm.