Fig 2: Characterization of donor Y40-ADSCs derived mitochondria. (a) (left) Mitochondrial distribution and representative transmission electron microscopy (TEM) image of intracellular mitochondria in Y40-ADSCs. Scale bar, 10 µm and 0.2 µm, respectively; (right) Images of isolated intact mitochondria. (b) Size distribution of isolated mitochondria. (c) Zeta potential of isolated mitochondria. (d) Western blot analysis of isolated mitochondria. TOMM20 and GLS are mitochondrial markers, RPL9 and ß-actin are cytoplasmic protein markers (three independent samples concentrated for one WB test). (e) Comparison of adenosine 5'-triphosphate (ATP) change during the storage of isolated mitochondria at 4°C and 37°C (n = 8). (f) Mitochondrial isolation efficiency from different cell number (cells/ml) (n = 3). Significantly different (one-way analysis of variance [ANOVA]): **p < 0.01 and ***p < 0.001

Fig 3: Treatment with RPL9 siRNA suppresses the abilities of invasion, migration and sphere formation in CR-CSCs.(A, B) Representative light microscopy images and relative percentage of invasive (A) and migratory (B) CD133+ HT29 CSCs treated with NC siRNA or RPL9 siRNA. The invaded cells were marked with red arrows in (A). Scale bar, 100 μm. (C) Representative light microscopy images and relative efficiency of sphere formation in CD133+ HT29 CSCs treated with NC siRNA or RPL9 siRNA. Scale bar, 100 μm. The difference in sphere forming capacity was compared by calculating size and quantifying number of spheres. *P < 0.05; **P < 0.01 vs. NC siRNA treatment. RPL9, ribosomal protein L9; CR-CSC, colorectal cancer stem cell; NC, negative control.

Fig 4: Targeting RPL9 inhibits the growth of CR-CSCs via disruption of ID-1 signaling axis.(A) Inhibition of growth of CD133+ HT29 CSCs by RPL9 silencing. (B) Reduction of CD133 and ID-1 mRNA expression in RPL9-silenced CD133+ HT29 CSCs. (C) Reduction of CD133, ID-1, and p-IκBα proteins in RPL9-silenced CD133+ HT29 CSCs. β-Actin was used as a loading control. ***P < 0.001 vs. NC siRNA treatment. RPL9, ribosomal protein L9; CR-CSC, colorectal cancer stem cell; ID-1, inhibitor of DNA-binding 1; NC, negative control.

Fig 5: Transfection of RPL9 siRNA reduces the metastatic potential of parental CRC cell population.(A, B) Representative light microscopy images and relative percentage of invasive (A) and migratory (B) HT29 and HCT116 cells treated with NC siRNA or RPL9 siRNA. Numbers of invaded and migrated cells were stained with crystal violet, counted, and graphed. Scale bar, 100 μm. (C) Detection of sphere formation in HT29 cells treated with NC siRNA or RPL9 siRNA. The differences in sphere forming capacity were compared by calculating both size and number of spheres. NC siRNA, negative control siRNA; RPL9 siRNA, RPL9-specific siRNA. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001 vs. NC siRNA treatment. RPL9, ribosomal protein L9; CRC, colorectal cancer; NC, negative control.