Fig 2: Characteristics of PGCCs. (A and B) Western blots of CD133, CyclinB1, CDC25C, EIF4A1, and CDK1 expression in TU212, cells after PTX withdrawal, as well as in PGCCs. (C and D) E-cadherin and vimentin expression in TU212, cells after PTX withdrawal, as well as in PGCCs

Fig 3: MiR-552-3p is highly expressed in GBC tissues and CSCs (n=41). (A) The relative miR-552-3p expression levels in GBC tissues and non-tumor adjacent tissues (NATs); (B) the relative expression level of miR-552-3p was detected in 41 patients; (C) the expression of miR-552-3p in spheroids and reattached cells was compared by q-PCR; (D) real-time PCR was used to detect the expression of miR-552-3p in passaged GBC spheroids; (E) real-time PCR was used to analyze the expression of miR-552-3p in CD44+ GBC cells sorted by flow cytometry relative to CD44- GBC cells; (F) real-time PCR was used to analyze the expression of miR-552-3p in CD133+ GBC cells sorted by flow cytometry relative to CD133- GBC cells. *, P<0.05. GBC, gallbladder cancer; CSCs, cancer stem cells; NATs, non-tumor adjacent tissues.

Fig 4: Honokiol down-regulates CD133 and Nestin expression via the JAK/STAT pathway in CD133 immunopositive cells. (A) CD133, Nestin and p-STAT3 levels were determined by western blot analysis. (B) Administration of 60 µM of honokiol, 20 µM of Stattic and mixture of both reduces p-STAT3, CD133 and Nestin levels. (C) Expression of CD133 (red) and Nestin (green) with nuclei counterstained with DAPI (blue). Scale bar: 20 µm. (D,E) Honokiol, Stattic and the mixture of both decrease numbers of U-87 MG cells immunopositive for CD133 and Nestin as evaluated by flow cytometry. Data are presented as means ± SEM from 3 independent experiments. ** p < 0.01 and *** p < 0.001 vs. vehicle control group.

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.