Fig 2: miR-140-3p attenuates the stem cell-like properties of LUAD cells. The percentage of CD133+ cells in (A) A549 and (B) Calu3 cells was analyzed by flow cytometry. (C) Representative images and the number of spheres with diameter >100 µm formed of A549 and Calu3 cells following transfection with miR-140-3p or the control mimic. mRNA expression levels of SOX2, OCT4, KLF4, NANOG and ABCG2 in (D) A549 and (E) Calu3 cells following transfection with miR-140-3p or the control mimic. Data are represented as the mean ± standard deviation from three independent experiments. *P<0.05 and **P<0.01 vs. control mimic. Scale bar, 100 µm. miR, microRNA; LUAD, lung adenocarcinoma; CD133, cluster of differentiation 133; SOX2, SRY-Box 2; OCT4, Octamer-binding transcription factor 4; KLF4, Kruppel like factor 4; ABCG2, ATP binding cassette subfamily G member 2; PE, phycoerythrin; FSC, Forward versus side scatter.

Fig 3: The effects of miR-3622a-3p and SALL4 on stemness features of CRC cells.a–d Flow cytometric analysis was used to examine the percentage of CD133(+) CRC cells. e–h Sphere formation assay was performed to investigate the stem-cell like properties of stable transfected CRC cells. i, j Expression levels of Sox2, Nanog, Oct4, CD44, and CD133 were determined by qRT-PCR in stable transfected CRC cells. k, l Protein levels of CSC-related biomarkers and pluripotency-related genes were detected by western blot in stable transfected CRC cells. All data are from three independent experiments and are presented as the means ± SD (*p < 0.05, **p < 0.01).

Fig 4: The cancer stem cells from glioma expressing high levels of glioma stem cell markers CD133 and ALDH1 along with TRPM7 and Notch1. (A) Morphologies of serum-free medium (SFM)-cultured stem-like tumorspheres from A172, U87MG, U373MG, and SNB19 glioma cells. (B–E) Flow cytometric analysis to measure CD133 expression in cells grown in monolayer and stem-like tumorspheres of A172 (B), U87MG (C), U373MG (D), and SNB19 (E). (F–I) ALDH1 activities determined by the Aldefluor assay in cells grown in monolayer and stem-like tumorsphere of A172 (F), U87MG (G), U373MG (H), and SNB19 (I). (J) The protein expression of TRPM7, Notch1, CD133, and ALDH1 determined by immunoblot and densitometry analysis with a fold change of each protein after normalization to individual loading control ß-actin (J).

Fig 5: CCL5 knockdown in TAMs suppresses prostate cancer growth, bone metastasis and PCSCs activity in vivo.a, b THP1-derived TAMs co-injection not only significantly accelerated PC-3-Luc xenografts growth but also increased the bone metastasis of prostate cancer xenografts, whereas CCL5 knockdown in THP1-derived TAMs could partly abrogate that. Mice were imaged using the IVIS Lumina XR in vivo imaging system to monitor PC3-Luc xenografts growth and bone metastasis (n = 12). Scale bar, 200 µm. c Immunohistochemistry indicated that CCL5 knockdown in THP1-derived TAMs could also significantly abrogate the up-regulation effect of TAMs on metastasis and stemness-related markers including ß-catenin, STAT3, CD133, and Vimentin in PC-3-Luc xenografts (n = 6). Scale bar, 50 µm. d CCL5 knockdown in THP1-derived TAMs could also significantly abrogate the promotion effect of THP1-derived TAMs on PCSCs self-renewal activity in vivo. Primary cells were isolated from fresh tumors by mechanical methods and subjected to PCSCs subpopulation analysis (n = 3). e In vivo limiting dilution assay was conducted to compare the tumorigenicity changes of PCSCs after CCL5 knockdown in co-cultured TAMs. The tumorigenic cell frequency estimate and p values were calculated by the limiting dilution analysis tool (http://bioinf.wehi.edu.au/software/elda/) (n = 8). *p < 0.05, **p < 0.01.