Fig 2: Coclaurine suppresses EFHD2 through inhibition of the transcriptional activity of FOXG1. (A) After cycloheximide (CHX, 10 mg/mL) treatment for 1 h, H1299 and A549 cells were treated or untreated with coclaurine (200 μM) for the indicated times. EFHD2 expression in each experimental group was determined by Western blot. (B) EFHD2 mRNA levels of H1299 and A549 cells treated or untreated with coclaurine (200 μM) for 24 h were determined by qPCR. GADHP and 18S rRNA served as internal controls for gene expression. (C) Schematic illustration showing the construction of the EFHD2 promoter sequence in the luciferase expression vector. (D) Relative luciferase activity with various EFHD2 promoter sequences in H1299 cells treated with coclaurine (200 μM) compared to the untreated control. (E) Schematic illustration showing the binding sites of transcription factors and the alteration of CDOCKER energy by coclaurine binding. (F,G) Three- and two-dimensional interactions between transcription factor FOXG1 (green), DNA (gray), and coclaurine (red) in two potential binding pockets. The binding amino acids in FOXG1 and DNA nucleotides with coclaurine are shown in the two-dimensional structures. (H) FOXG1 thermal stability in H1299 cells treated or untreated with coclaurine (200 μM) at the indicated temperatures was determined by Western blot. The relative FOXG1 level (FOXG1/actin) was normalized to the unheated control of each group. (I) FOXG1 and EFHD2 expression and (J) cell viability of H1299 cells with or without FOXG1/KD in the presence or absence of coclaurine (200 μM) were determined by Western blot and MTT assay, respectively. Data are displayed as the means ± SD. For statistical analysis, a 2-tailed unpaired Student’s t-test (J). ★★, p < 0.01.

Fig 3: Representative immunocytochemistry images of BR1, NSC (p.2), and NSC (p.8–9), highlighting the expression of NSC‐associated proteins and corresponding quantification graphs. Quantitative analyses were performed within the same passage‐specific experimental framework, with fluorescence intensities normalized to total cell number. (a) Brightfield images show cell morphology; fluorescence images indicate marker expression. (b) Mean cell counts per field were similar across groups. (c) SOX1 labeling was most intense in NSCs (p.8–9). (d) SOX2 labeling was higher in BR1 compared to NSC (p.2). (e) PAX6 was expressed in all groups, with no statistical differences. (f) NESTIN expression was strongest in NSCs (p.8–9). (g) FOXG1 was predominantly expressed in NSCs (p.2). [BR1, white bars; NSC (p.2), medium gray; NSC (p.8–9), dark gray]. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (p.2) passage 2, (p.8–9) passages 8 and 9. One‐way ANOVA with Tukey's post hoc test. n = 5 images per group. Scale bars: 100 µm.