Fig 2: Schematic image of CD133 functions in cells. Under preferential growth (high nutrients) conditions, CD133 is phosphorylated in its intracellular C-terminal domain by Src family tyrosine kinases. As a result, phosphorylated CD133 activates a p85 subunit of phosphoinositide 3-kinase (PI-3K) by binding, and PI-3K, in turn, activates downstream targets, such as Akt, thereby promoting cell proliferation. Autophagic activity is at its basal level. On the other hand, under severe growth (low nutrients) conditions, non-phosphorylated CD133 is transported from the plasma membrane to the pericentrosomal region through endocytosis. After endocytosis, HDAC6 and dynein motors assist CD133 endosome trafficking along microtubules to centrosomes. Endosomal CD133 localised in a pericentrosomal region captures GABARAP to inhibit the GABARAP-ULK1 interaction, thereby suppressing the initiation of autophagy. Endosomal CD133 is a recycling endosome that may be mediated through the functions of Rab11. Thus, pericentrosomal CD133 has the unique property of maintaining a cell undifferentiated status by inhibiting autophagy.

Fig 3: Effect of USP18 on chemotherapy sensitivity of colorectal cancer cells. a–f Cellular chemotherapy sensitivity experiment on three common chemotherapeutics (fluorouracial, doxorubicin and cisplatin) in USP18 knocked-down SW480 cells and USP18 over-expressed DLD1 cells. g–h Western blot and qRT-PCR analysis of stem cell-related proteins (CD133/CD44). *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: Inhibition of KLF5 decreases spheroid growth in EOC cells. (A, B) Isolation of spheroid-forming cells from EOC cells. Sphere forming assay was performed by culturing EOC cells (5 × 102 cells/well) in sphere medium for 14 days in 24-well ultra-low attachment plates. Proteins were isolated from spheroid-forming cells and respective parental adherent cells and immunoblotted with antibodies against KLF5, pSTAT3, STAT3, CD44, CD133, NANOG, OCT4 and GAPDH. (B, C) Silencing of KLF5 inhibits self-renewal ability of spheroids. EOC cells were transfected with KLF5 shRNA and cells were subjected to sphere forming assay. Spheroids in the entire well were counted. (D) Silencing of KLF5 inhibits stemness of spheroids as confirmed by immunoblotting using stem cell markers. EOC cells were transfected with scramble or KLF5 shRNA’s and grown in sphere medium. Proteins were isolated from spheroids and immunoblotted with antibodies against KLF5, pSTAT3, STAT3, CD44, CD133, NANOG, OCT4 and GAPDH. Data presented in the bar graphs are the mean ± SD of triplicates in an independent experiments which was repeated for at least two times with the same results. *Indicates a statistically significant difference compared to control with p < 0.05. Western blot experiments were repeated at least two times with the same results.

Fig 5: BJJP and BMSCs suppress CSCs cell proliferation and enhance cell apoptosis through regulating miR-140. (A) Transfection efficiency was verified by RT-qPCR; (B, C) RT-qPCR and western blot were applied to detect the expression of CD24, CD133 and EpCAM after cell transfection; (D) cell proliferation ability was assessed by cell clone formation; (E) cell viability was measured by CCK-8 assay; (F) cell apoptosis was determined by Flow cytometry. N=3, *, vs control group, p<0.05; **, vs control group, p<0.01; #, vs BJJP group, p<0.05; *, vs BMSCs group, p<0.05; *, vs BJJP＋BMSCs group, p<0.05; EpCAM, epithelial cell adhesion molecule; CSCs, cancer stem cells; BMSCs, bone mesenchymal stem cells; BJJP, Bie Jia Jian pill.