Fig 2: The effect of ZSCAN18 overexpression in vivo. (A) Tumor implant size. Non: non-transduced LN-229 cells. ZSlo: LN-229 cells transduced with control lentivirus. ZSov: LN-229 cells transduced with ZSCAN18-encoding lentivirus. (B) Tumor implant weight 28 days after subcutaneous implantation. (C) Exclusion of H-2Dd-positive mouse cells and gating of H-2Dd-negative LN-229 cells by flow cytometry. (D and E) Ki67 staining in LN-229 cells sorted from tumor implants. Representative flow cytometry histograms are illustrated in (D). Statistics of the frequencies of Ki67+ cells are shown in (E). (F and G) Protein levels of indicated markers in LN-229 cells sorted from tumor implants. Representative blots are shown in (F), and densitometry results are shown in (G). The numbers above the bars indicate fold increases or decreases in indicated protein levels. (H and I) TMZ-induced apoptosis and necrosis of sorted LN-229 cells. Representative plots are illustrated in (I). Statistics of the frequencies of apoptotic and necrotic cells are shown in (I). n = 4 to 8 per group. *: p < 0.05; **: p < 0.01; ***: p < 0.001. One-way ANOVA.

Fig 3: Cell migration and drug after ZSCAN18 overexpression. (A & B) Apoptosis and necrosis of LN-229 cells after 100 μM TMZ treatment. Non: non-transduced LN-229 cells. ZSlo: LN-229 cells transduced with control lentivirus. ZSov: LN-229 cells transduced with ZSCAN18-encoding lentivirus. Representative plots are illustrated in (A). Statistics of the frequencies of apoptotic and necrotic cells are shown in (B). (C) LN-229 viability after treatment with indicated concentrations of TMZ. (D) Migrated LN-229 cell number after 12-h and 24-h incubation in Transwell plates. n = 5 or 6 per group. ***: p < 0.001. One-way ANOVA.

Fig 4: The significance of GLI1 to ZSCAN18-mediated effects. (A) mRNA levels of indicated sonic hedgehog signaling components in LN-229 cells. Non: non-transduced LN-229 cells. ZSlo: LN-229 cells transduced with control lentivirus. ZSov: LN-229 cells transduced with ZSCAN18-encoding lentivirus. The numbers above the bars indicate fold decreases in the levels of indicated proteins. (B) GLI1 protein in LN-229 cells. The blots represent two independent experiments. (C) GLI1 protein in LN-229 cells on day 2 after secondary lentiviral transduction. ZSlo: ZSlo cells without secondary transduction. ZSov-Glo: ZSov cells transduced with secondary control lentivirus. ZSov-Gov: ZSov cells transduced with GLI1-encoding lentivirus. (D) Growth curves of LN-229 cells. *: p < 0.05; **: p < 0.01; ***: p < 0.001 between the ZSlo group and ZSov-Gov group. ##: p < 0.01; ###: p < 0.001 between the ZSov-Glo group and ZSov-Gov group. (E) LN-229-derived sphere number at passage 1 (P1), passage 2 (P2), and passage 3 (P3). (F & G) Apoptosis and necrosis of LN-229 cells after 100 μM TMZ treatment. Representative plots are illustrated in (F). Statistics of the frequencies of apoptotic and necrotic cells are shown in (G). n = 5 or 6 per group. ns: not significant. One-way ANOVA.

Fig 5: LN-229 cell self-renewal after ZSCAN18 overexpression. (A) LN-229 cell-derived sphere number at passage 1 (P1), passage 2 (P2), and passage 3 (P3). Non: non-transduced LN-229 cells. ZSlo: LN-229 cells transduced with control lentivirus. ZSov: LN-229 cells transduced with ZSCAN18-encoding lentivirus. (B) Diameters of LN-229 cell-derived spheres. (C & D) Protein levels of indicated markers in P3 spheres. Representative blots are shown in (C), and statistics are shown in (D). The numbers above the bars indicate fold increases or decreases in indicated protein levels. n = 4 to 8 per group. *: p < 0.05; **: p < 0.01; ***: p < 0.001. One-way ANOVA.