Fig. 7: LPA-PKD-1 signaling pathway in self-renewal and plasticity of breast cancer stem cells.

a Induction of ALDH1A1 expression in BC cells (upper panel). The E0771 cell was cultured in RPMI-1640 medium with 10% FBS and 1% penicillin/streptomycin. The following starvation in serum-free medium for 6 h, the cells were treated with LPA (10 μM) for 24 h, the cell lysates were collected for Western Blot. Duplicated experiments were performed, and shown is a representative result; ALDH1A1 expression in implanted E0771 BC (lower panel). Immunofluorescence microscopy was used to detect ALDH1A1⁺BC cells in tumor tissues. Representative images are shown. Scale bar = 50 µm. b MCF-7 cells were exposed to 10 µm LPA, 1 µm CRT0066101 (PKD inhibitor), 1 µm Ki16425 (LPA_1,3 antagonist), and their combinations for 24 h. The control and treated MCF-7 cells were incubated with ALDH1A1 antibody followed by an appropriate secondary antibody. Representative images are shown from triplicate experiments. Fluorescence intensity was measured by ImageJ and calculated by the corrected total cell fluorescence (CTCF). The relative expression was shown as the mean ± SEM. ^**P < 0.01 compared with the control. Scale bar = 50 µm. c Total RNA was extracted from the MCF-7 control, those transduced with wild-type PKD-1 (PKD-WT) or the PKD-1-transduced MCF-7 cells that were treated with PKD inhibitor CRT0066101. The mRNA levels were assayed by RT-qPCR. ^*P < 0.05 compared with the control. d MCF-7 cells transduced with wild-type PKD-1 (PKD-WT) were stained with Notch 1 antibody, followed by an appropriate secondary antibody. The fluorescence intensity was measured by ImageJ and calculated by the CTCF. Representative images are shown. Scale bar = 50 µm. In addition, the total RNA was collected from the control and MCF-7 with overexpressing PKD-WT were collected and Notch1 mRNA levels were assayed with RT-qPCR. Triplicate experiments were performed and results shown as the mean ± SEM. ^**P < 0.01 or ^***P < 0.001 compared with the control. e Overexpression of PKD-WT increased protein expression of CD44 in BC cells. MCF7 cells were transduced with PKD-WT and protein lysate was collected for Western blots. MCF7 without PKD-WT or 293 T cells transduced with PKD-WT as an experimental control. f Overexpression of PKD-WT induced KLF4 expression in BC cells. MCF7 cells transduced with PKD-WT were starved in serum-free DMEM media overnight and treated with/without CRT0066101 (0.5 µm) for 24 h. Total RNA was extracted for the detection of KLF4 mRNA levels by RT-qPCR. *P < 0.05 or ***P < 0.001. g. MCF7 cells were serum-free starved overnight and then treated with LPA (10 µm) and/or LPA antagonist Ki16425 (1 µm) or CRT0066101 (1 µm) for 24 h. The cell lysates were collected for protein expression by Western Blot. Duplicate experiments were performed.