Fig 2: Silencing TLN1 promotes apoptosis and inhibits tumour growth of MDA-MB-231 cells.(A) TLN1 expression in the indicated breast cancer cell lines, using western blotting. (B) The efficiency of TLN1 silencing with shRNA in MDA-MB-231 cells was evaluated by western blotting. (C) Silencing TLN1 inhibited the proliferation of MDA-MB-231 cells (scale bar, 20 µm, p < 0.05 at 48 hr and p < 0.01 at 72 hr). (D) Silencing TLN1 enhanced spontaneous apoptosis in MDA-MB-231 cells, which was detected by Flowcyto. (E) Silencing TLN1 increased apoptosis-related vacuoles (red arrows), which was detected by transmission electron microscopy (TEM). (F) Silencing TLN1 reduced the growth of xenografted triple-negative breast cancer (TNBC) tumours in NOD/SCID mice (n = 8 per group). Data are presented as representative images, flow cytometric plots, or the mean ± standard error of the mean (SEM) of each group from three separate experiments. *p < 0.05, **p < 0.01 vs. the NC group. Figure 2—source data 1.Silencing TLN1 promotes apoptosis and inhibits tumour growth of MDA-MB-231 cells.

Fig 3: PDGFR-ß interacts with FAK and TLN1 in ECs. a Strategy for identifying PDGFR-ß binding proteins using LC-MS/MS analysis. Bone morrow endothelial cells (BMECs) treated with PDGF-BB (300 ng·mL-1) for 24 h were subjected to immunoprecipitation (IP) with an antibody against PDGFR-ß followed by LC-MS/MS analysis. b The PDGFR-ß binding proteins FAK and TLN1 were identified with LC-MS/MS analysis. c PDGFR-ß was immunoprecipitated from BMECs with an anti-PDGFR-ß antibody. The presence of FAK, TLN1, and PDGFR-ß in these immunoprecipitates was evaluated with immunoblotting. Western blot analysis of FAK, TLN1, and VEGF in the BMECs treated with different PDGF-BB doses (0-300 ng·mL-1) (d) or for different times at 0 h–36 h (e). Western blot analysis of FAK, TLN1, and VEGF in the BMECs treated with FAK inhibitors (f) or TLN1 siRNA (g)

Fig 4: CREPT expression influences actin filament/focal adhesion formation-related signaling molecules in BLM cells. (a) Western blotting was performed to evaluate expression of RhoA, Diaphanous related Formin 1 (mDia1), FilaminA, focal adhesion kinase (FAK), Talin-1, Paxillin, Tensin 2. Beta-actin (ACTB) was used as the loading control. Representative results are shown followed by mean protein expression values from four independent experiments. (b) Activated RhoA levels were quantified with small GTPase activation assay, and relative values are shown as mean ± SEM of three independent experiments. ** p < 0.01, *** p < 0.001.

Fig 5: Effects of bupleuri radix water extract (W-BR) and bupleuri radix ethanol extract (E-BR) on proliferation, adhesion, and migration of HaCaT cells. (A) Prediction of indications of W-BR. The ranking of diseases associated with W-BR was measured as the number of common pathways that were significantly enriched in expression signatures of disease and W-BR. (B) HaCaT cells were treated with the indicated concentrations of W-BR or E-BR for 24 h, and then cell viability was measured. (C) HaCaT cells treated with or without W-BR and E-BR were added onto collagen I-coated wells. After incubating cells for 30 min, attached cells were quantitated. (D) The levels of ?-actinin, focal adhesion kinase, talin-1, and vinculin were detected by immunoblotting in W-BR or E-BR-treated HaCaT cells. Relative band intensities were calculated after normalization to ß-actin expression. Data are representative of two independent experiments. (E) HaCaT cells were pretreated with W-BR or E-BR for 24 h and evaluated for migration ability across the transwell membrane. Scale bar = 200 µm. Original magnification × 100. (F) Wound healing in W-BR or E-BR-treated HaCaT cells was detected using the RadiusTM 96-well Cell Migration Assay kit. Images were taken at the indicated timepoints, and the wound area is indicated with a solid line. Data are expressed as means ± standard deviation from triplicate samples. *p < 0.05 and **p < 0.01 vs. vehicle-treated control. E-BR: bupleuri radix ethanol extract; W-BR: Bupleuri radix water extract, FAK: focal adhesion kinase.