Fig 2: Baicalin upregulated DEPP expression and induced senescence in colon cancer cells in vivo.a, b Effects of baicalin on tumor volume and body weight. The data are representative of results obtained from 5 mice. c Tumor sections were subjected to hematoxylin and eosin staining, SA-β-Gal staining and IHC for Ki67, p16INK4A and cleaved-caspase 3. Original magnification was 400×. Every single shown picture is representative of five sections from every group. Different sections represent different tumors. The representative images and the calculation of the percentage of positive cells are shown in d–g; the data are the means ± SD (***P ≤ 0.001). h Tumors were collected for western blot analysis of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16INK4A, pRb, Rb and β-actin. Every single lane represents one tumor from an individual mouse. i Tumor sections were subjected to cleaved-caspase 3. Original magnification was 400×

Fig 3: Ras/Raf/MEK/ERK signaling pathway was involved in DEPP-mediated tumor cellular senescence in colon cancer cells.HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with siRNA targeting Raf1 and ERK, then immunoblotting analysis a and SA-β-Gal staining c were carried out. Shown are representative of 4 experiments. HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with Sorafenib or U0126 for 24 h, then immunoblotting analysis b, SA-β-Gal staining d, EdU staining e and colony formation assay f were carried out. Shown are representative of 4 experiments. The quantification of SA-β-Gal and crystal violet staining represents the means ± SD. The integrated optical density of EdU staining represents the means ± SD. (***P ≤ 0.001 versus the control group). g Immunoblotting of Flag-DEPP immunoprecipitates from HCT116 cells with or without the treatment with baicalin or siDEPP. The binding of DEPP to Ras in cells is presented relative to that in the untreated cells. Shown are representative of three experiments

Fig 4: Crabp1-selective compound C3 enhances Crabp1 interaction with the Raf-RBD and competition with active Ras for RBD binding. (A) In vitro interaction assay. Compound C3 increases Crabp1 interaction with the Raf-RBD. (B) Compound C3/Crabp1 reduces active Ras interaction with the Raf-RBD. (C) HEK293T cells reconstituted with the indicated expression vectors and treated with, or without, C3 for 30 min were monitored for Raf activation in cells, indicated with p-cRaf (S338).

Fig 5: Detection results of MAPK pathway-related proteins in breast cancer tumor tissues.Notes: (A–L) The WB image and relative protein expression of p-RAF1, p-MEK, p-ERK, p-P38, Bax and Bcl-2. Data are presented as mean ± SD of three experiments, n=3. (#P < 0.05, ##P < 0.01, vs model group).