Fig 2: ALDH1A1 immunostaining of original ESCC cell lines and curcumin-surviving lines. The histograms summarize cell counts according to staining intensity as shown in Figure. 4. The maxima of the high-staining subpopulations in the original cell lines are shown by the asterisk. Curcumin-surviving cell lines, indicated with “S”, showed a significant loss of the high-staining subpopulation. The mean intensity of ALDH1A1 expression was significantly different among the original lines (F=307.047, p<0.0001). KY-5 staining was significantly higher than all other lines. YES-2 was significantly higher than YES-1, TE-8, TE-1, and KY-10. Also, TE-8 was significantly higher than YES-1, TE-1 and KY-10. The mean intensity of ALDH1A1 expression levels was also significantly different among the surviving lines (F=611.440, p<0.0001). KY-5S staining was significantly higher than all other surviving lines. YES-2S was significantly higher than TE-8S, KY-10S, TE-1S and YES-1S. TE-8S was higher than KY-10S, YES-1S and TE-1S. Also, KY-10S was significantly higher than YES-1S and TE-1S. The mean intensity of high-staining cells in the original cell lines was 477.80 ± 93.82, 243.77 ± 33.54, 222.23 ± 42.02, 199.70 ± 39.31, 180.88 ± 28.31, and 149.67 ± 16.29 for KY-5, YES-2, TE-8, KY-10, YES-1, and TE-1, respectively. The mean intensity of high-staining cells in the surviving cell lines was 496.93 ±108.38, 234.95 ±28.23, 206.67 ±26.77, and 145.55 ± 24.52 for KY-5S, YES-2S, KY-10S, and YES-1S.

Fig 3: TGF-ß treatment induced EMT and the stemness of glioma cells in a concentration-dependent manner. (A–C) The mRNA and protein level of E-cadherin and Slug were examined in glioma cells treated with different concentrations of TGF-ß as indicated. (D) The capacity for cells spheroid formation was determined on the same glioma cells analyzed in (A). (E) ALDH1 activity was measured on the same glioma cells analyzed in (A). (F–H) The mRNA and protein levels of ALDH1 and Nanog were determined in cells depicted in (A). Scale bar, 100 µm. The difference was assessed using one-way ANOVA with the Tukey–Kramer post hoc test. Data are presented as the mean and SD; n = 3, *P < 0.05, **P < 0.01 vs Control. The densitometric analysis values are the means of three independent blots and representative blots are shown.

Fig 4: THOR positively regulates the stemness of OS cells. (A) Representative picture of spheroids formed by MG63 cells. (B) mRNA levels of stemness markers (Nanog and ALDH1) were examined in MG63 adherent cells and spheroids. (C) ALDH1 activity was determined in MG63 adherent cells and spheroids. (D) THOR level was detected in MG63 adherent cells and spheroids. (E) The infection efficiency of Lenti-THOR and Lenti-THOR-knockdown was evaluated in MG63 adherent cells and spheroids, respectively. (F) mRNA levels of stemness markers (Nanog and ALDH1) were examined in MG63 cells with or without THOR overexpression. (G) mRNA levels of stemness markers (Nanog and ALDH1) were measured in MG63 spheroids with or without THOR knockdown. (H) Protein levels of stemness markers (Nanog and ALDH1) were determined in cells and spheroids described in (F,G). (I) ALDH1 activity was determined in cells and spheroids depicted in (F,G). (J,K) Spheroid formation ability was evaluated in cells and spheroids described in (F,G) through measuring the spheroids size (J) and number (K). Difference was assessed using one-way ANOVA with the Tukey–Kramer post hoc test. Data are presented as the mean ± SD, n = 3, **P < 0.01 vs Control. Scale bar, 50 µm.

Fig 5: Apatinib inhibited tumor growth in vivo.a Male BALB/c nude mice (5 weeks of age) were injected with 5 × 106 A549 cells. After 14 days, apatinib (100 mg/kg body weight) was orally administrated daily for another 14 days. b Subcutaneous tumors in mice on the 28th day after the injection of A549 cells. c–d Tumor volumes and masses were determined on the 28th day after injection. Unpaired t-test was used. Data are presented as mean ± SD (n = 5). **p < 0.01 compared to the control group. e H&E staining of tumor, heart, liver, spleen, lung and kidney. Scale bar = 50 µm. f Immunohistochemical staining of CD133, ALDH1A1, ABCG2, ß-catenin, SOD2, and GPX4 expression in the xenograft tumors derived from the control group and the apatinib-treated group. Scale bar = 50 µm. Unpaired t-test was used. Data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01 compared to the control group. g The representative working model of the study. Apatinib directly inhibited lung CSC-like properties by targeting Wnt/ß-catenin signaling and decreasing lung CSC-specific markers; apatinib disturbed redox balance to induce ROS generation; ROS production and ß-catenin suppression interacted with each other, leading to the inhibition of lung CSC stemness.