Fig 2: Development of the heal.immune score and validation of its prognostic value.(A) Flow chart of the procedure to develop the heal.immune risk score. Via stepwise Cox estimation, six genes were chosen as a gene signature to calculate the heal.immune risk score. (B) The expression levels of the risk score genes in HCC. (C) The correlation of the six genes; red indicates ICGs and blue indicates wound healing-related genes. (D) The heal.immune scores of C1 and C2. (E–F) Multivariate Cox estimation of the six genes as OS risk factors in meta-cohort (validation group) (E) and another independent Fudan cohort (F). (G–H) Kaplan-Meier curve of showing the OS between heal.immune score high group and low group in meta-cohort (validation group) (G) and another independent Fudan cohort (H). (I) The represent images of immunohistological staining (FCER1G, CD86, CD40, ITGA5, TPA, CyclinB1) in heal.immune score high group or low group. HCC, hepatocellular carcinoma; ICG, immune checkpoint-related gene; CD40, Tumor Necrosis Factor Receptor Superfamily Member 5ECM, extracellular matrix; CD86, B-Lymphocyte Activation Antigen B7-2; FCER1G, Fc Fragment Of IgE Receptor Ig; ITGA5, Integrin Subunit Alpha 5.

Fig 3: C. butyricum RH2 treatment reversed CFSS-induced upregulation of PAI-1 in the hippocampus. (A–C) Western blot analysis of expression of PAI-1 and tPA in the hippocampus of CFSS rats before and after C. butyricum RH2 treatment. (D–E) The immunoreactive spots of PAI-1 and tPA protein in the hippocampus of CFSS rats before and after C. butyricum RH2 treatment. *p < 0.05, **p < 0.01 versus sham group; #p < 0.05, ##p < 0.01 versus stress group; ^p < 0.05, ^^p < 0.01 versus stress + C. butyricum group.

Fig 4: Bmal1 deficiency augments the PAI-1-tPA/uPA-plasmin axis. A) The stromal vascular fraction (SVF) of CRC tumors grown in Bmal1+/+ and Bmal1-/- mice were isolated by FACS sorting. RNAs were extracted and subjected for sequencing. B) Volcano plot presenting differential gene expression in isolated SVF from Bmal1+/+ versus Bmal1-/- mice. Orange indicates significantly increased genes and blue indicates significantly decreased genes. C) Genome-wide expression profiling of SVF from Bmal1+/+ and Bmal1-/- tumor tissues (n = 4 samples per group). Serpine1 was identified as one of the highly down regulated genes. D) Quantification of Serpine1 mRNA level in CRC tumors, SVF, and FACS-isolated FAP+ CAFs from Bmal1+/+ and Bmal1-/- tumors (n = 3 samples per group). E) Schematic diagram of Serpine1 transcription. The Serpine1 is a direct transcriptional target of BMAL1-CLOCK heterodimers and the Serpine1 gene promoter contained canonical E-boxes of BMAL1-CLOCK binding sites. F–H) Relative PAI-1, uPA, and tPA protein levels and quantification in CRC tumors, SVF, and CAFs from Bmal1+/+ and Bmal1-/- mice (n = 4 samples per group). I) ELISA quantification of active PAI-1 in CRC tumors, SVF, CAF supernatant and plasma from tumor-bearing mice (n = 5 samples per group). J,K) Quantification of uPA and tPA activity in CRC tumors, SVF, and CAF supernatant from Bmal1+/+ and Bmal1-/- mice (n = 4 samples per group). L) Quantification of plasmin activity in CRC tumor and plasma from tumor-bearing mice (n = 6 samples per group). Data presented as mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed student t-test.

Fig 5: Activation of TGF-ß signaling in Bmal1-/- tumor and CAFs. A) ELISA quantification of active TGF-ß in CRC tumors and plasma from tumor-bearing mice (n = 4 samples per group). B,C) Immunoblot and quantification of p-SMAD2 and a-SMA in CRC tumor and CAFs from Bmal1+/+ and Bmal1-/- mice (n = 4 samples per group). D) Cell motility and quantification (n = 5 colonies per group). Quantification of CAFs number after 48 h from Bmal1+/+ and Bmal1-/- mice (n = 4 random fields per group). E) Schematic diagram of activation of TGF-ß signaling. Tumor and tumor-bearing host plasma provide resource of latent TGF-ß. Bmal1 deficiency augment PAI-1-uPA/tPA-plasmin axis. High plasmin serves as activator convert latent TGF-ß into active form. Data presented as mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed student t-test.