Fig 2: Reanalysis of clinical scRNA-seq with CSC-like signatures. (A) Flowchart representing the strategy of reanalysis using public scRNA-seq datasets. We downloaded GSE161529, GSE176078 and GSE180286 including scRNA-seq data from 19 TNBC patients. The integrated datasets were analyzed with Seurat. Log normalization, scaling, principal component analysis (PCA) and UMAP visualization were performed following the basic protocol in Seurat. To extract the cancer cells, cells expressing EPCAM/KRT8 (epithelial marker) were filtered. Cancer cells (48,362 cells) were extracted from 107,164 single cells. (B) UMAP plot of cancer cells from 19 TNBC patients with sample IDs. (C,D) Expression analysis of CD44 (C; expression level>2) and HMGA1 (D; expression level>2) with UMAP plots. (E) UMAP plot of CD44-high, HMGA1-high, HMGA1/CD44-high and marker-negative cancer cells. (F) The proportion of cancer cells that expressed CD44 and HMGA1 are represented in a bar plot.

Fig 3: Detection of the molecular expression in SK-BR-3, MCF-7, and LD breast cancer cell lines. (A-C) TGFBR2, Smad3, and CD44 expression levels in SK-BR-3, MCF-7, and LD cells were measured by RT-qPCR analysis after miR-7 mimic transfection. (D-F) TGFBR2, Smad3, and CD44 relative expression levels in SK-BR-3, MCF-7, and LD cells were detected after the inhibitor transfection by RT-qPCR. *P<0.05, **P<0.01 and ***P<0.001. TGFBR2, transforming growth factor-ß receptor 2; RT-qPCR, reverse transcription-quantitative polymerase chainreaction; miR-7, microRNA-7.

Fig 4: ALDH1A3 knockdown reduces CD44 expression in MDA-MB-231 cells and their proliferation in BCSCs. (A) Detection of miR-7 expression after ALDH1A3 knockdown in MDA-MB-231 cells by RT-qPCR. (B) Analysis of the effectiveness of ALDH1A3 knockdown in MDA-MB-231 cells. (C) Demonstration of transfection efficiency with lenti-miR-7, miR-7 mimic and miR-7 inhibitor. (D) Overexpression of miR-7 decreased CD44 gene expression. (E) siALDH1A3 reduced the ratio of CD44+ cells analyzed by FCM. (F) Statistical analysis revealed that the CD44+ cell average fluorescence intensity was reduced. (G) The proliferation of BCSC-siALDH1A3 cells was evaluated with FCM assay. (H) Statistical analysis of the cell cycle of BCSCs. *P<0.05, **P<0.01 and ***P<0.001. ALDH1A3, aldehyde dehydrogenase 1 family member A3; BCSCs, breast cancer stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR-7, microRNA-7; si, small interfering; FCM, flow cytometry.

Fig 5: Bufalin antagonizes cisplatin with regard to the expression of stemness markers in colorectal cancer cells in vitro. (A) All secondary tumorspheres are dissociated into single cells and are seeded on cover slips in a 48-well plate. The protein expression of CD133, CD44, OCT4, SOX2, NANOG, and ABCG2 are evaluated by immunofluorescence. After 14 days, images of tumorspheres were obtained using microscopy. (B) The protein expression of CD133 and CD44 was evaluated using a flow cytometry histogram plot. (C) The protein expression of CD133, CD44, OCT4, SOX2, NANOG, and ABCG2 was evaluated and normalized with GAPDH using western blotting.