Fig 2: Macrophages stimulated with CRX555 show delayed NF-?B nuclear translocation in comparison to cells stimulated with LPS.RAW264.7 macrophages stably expressing RelA-EGFP and a TNFa promoter-mCherry reporter were stimulated with LPS or CRX555. Confocal time-lapse images were captured every 3 min for 15 hr. (A) Stills of NF-?B nuclear translocation and mCherry expression following 2 nM LPS stimulation. (B) NF-?B dynamics was assessed as the ratio of the nuclear:cytoplasmic EGFP fluorescence. Time to the first peak of NF-?B translocation was measured in response to LPS or CRX-555. (C) Fold increase of mCherry intensity following stimulation with LPS or CRX555 (the dotted line indicates no increase in mCherry). Each dot represents a single cell from four independent experiments (n = 30–50 for each condition). The bar represents mean value ± s.d. ***p<0.0001, **p<0.01, *p<0.05, one-way ANOVA with Kruskal- Wallis post-test, compared with cells stimulated with the lowest concentration of LPS (0.002 nM). (D and E) Statistical analysis of the correlation between the speed of NF-?B nuclear translocation and TNFa promoter activation. Spearman’s correlation coefficient was used to assess links between time to the first peak of NF-?B nuclear translocation and the fold increase in mCherry intensity or magnitude of NF-?B translocation following LPS (D) (F) or CRX-555 stimulation (E) (G).

Fig 3: Effects of cbFeD and oxaliplatin on growth of xenograft tumors. Nude mice were subcutaneously injected in the right flank with 2.0×106 SW480 cells in 0.1 ml PBS. Once tumors had formed, tumor volume was measured. The mice were then randomly divided into three groups (n=5): Normal control group, mice treated with normal saline via gavage; high cbFeD group, mice treated with a high dose (20 g/kg) of cbFeD via gavage; oxaliplatin group, mice with colon cancer treated with oxaliplatin (5 mg/kg) via gavage. After 6 weeks, the dissected tumors were collected and H&E and IHC staining were performed. (A) Tumor size. (B) tumor volume. (C) H&E staining of xenograft tumors. Magnification, ×400. (D) IHC staining of p65, LC3B-II and Beclin-1. Magnification, ×400. **P<0.01 and ###P<0.001 vs. control. cbFeD, Codonopis bulleynana Forest ex Diels; LC3, microtubule-associated protein 1 light chain 3; IHC, immunohistochemistry.

Fig 4: Effects of PDTC and a high dose of cbFeD on expression levels of p65, I?Ba, p-I?Ba, LC3B-I, LC3B-II and Beclin-1, and on autophagic vacuoles in HCT116 and SW480 cells. Cells were treated with the p65 inhibitor PDTC, with or without, cbFeD-containing serum solutions prepared from mice treated by gastrogavage with saline (control), or 20 (high) g/kg of cbFeD. (A) Expression levels of p65, I?Ba, p-I?Ba, LC3B-I, LC3B-II and Beclin-1 were examined using western blot analysis and (B) detection of autophagic vacuoles was performed using AO staining. cbFeD, Codonopis bulleynana Forest ex Diels; PDTC, pyrrolidine dithiocarbamate; NF-?B, nuclear factor-?B; I?B, inhibitor of NF-?B; p-, phosphorylated; LC3, microtubule-associated protein 1 light chain 3; AO, acridine orange.

Fig 5: Immunofluorescence images of p65 in HCT116 and SW480 cells. Cells were treated with cbFeD-containing serum solutions prepared from mice treated by gastrogavage with saline (control), or 5 (low), 10 (mid) or 20 (high) g/kg of cbFeD, following by immunofluorescence staining of p65 (magnification, ×200). Green, p65; blue, DAPI; Merge, p65+DAPI. cbFeD, Codonopis bulleynana Forest ex Diels.