Fig 2: HEMF cell line mRNA expression and protein secretion. (A) mRNA expression of upregulated genes identified by RNA sequencing was evaluated in a HEMF cell line similarly treated with acidic bile salts and compared to untreated cells (grown in neutral serum-free myofibroblast media (SFMM; pH 7.2). mRNA expression of the following differentially expressed genes were evaluated: CXCL8, IL-6, CXCL3, CCL5, CXCL6, AREG, EREG, and VEGFA. Statistically significant differences are shown by * symbol with p value = 0.05. Results shown represent the means ± SEM of three independent experiments conducted with HEMF cell line. (B) Proteins of genes identified by RNA sequencing were evaluated in supernatant of untreated and acidic-bile-salt-treated HEMFs. Statistically significant differences are shown by * symbol with p value = 0.05. Results shown represent the means ± SEM of three independent experiments conducted with HEMF cell line.

Fig 3: Model summarizing the pathway described in this study along with known effects of CCL20 and CXCL8 signaling in the context of cancer.Some of the relationships in this linear pathway may be the result of indirect mechanisms (indicated by dotted line) and/or also include the contribution of additional co-factors (not shown).

Fig 4: C/EBPd is sufficient to induce CXCL8 and CCL20 transcription.a qRT-PCR analysis of CXCL8 and CCL20 mRNA levels in MDA-MB-435S cells transfected with siSCR or siCEBPD, treated with DMSO (-) or Tg (100 nM) for 6 h and with or without with GSK-414 (1 µM) pre-treatment as indicated. b, c qRT-PCR analysis of CXCL8 and CCL20 mRNA levels in MDA-MB-435S (a) and HEK293T (b) cells transfected with expressing constructs of full length (WT) or transactivation domain-truncated (?TA) C/EBPd with FLAG-tag. Empty vector was used as control. Panels on the right show Western blots to demonstrate expression of ectopic C/EBPd proteins. d Schematic of the CXCL8 gene along with the C/EBPd ChIP-Seq track from HepG2 cells as reported by the ENCODE database, and the sequence between positions -110 and -1 harboring a peak and C/EBP motifs (shaded) and indicating the position (-65) of the deletion mutation of the reporter construct shown in e. e Luciferase reporter assay in HEK293T cells co-transfected with C/EBPd expression constructs as in b and Firefly luciferase (FLuc) reporter constructs containing the proximal CXCL8 promoter region from position -110 or -65 to -1. f Western blot analysis of representative extracts from e showing FLuc and C/EBPd (anti-FLAG) protein expression. g qRT-PCR analysis of the mRNA levels of CEBPD, CXCL8, and CCL20 in SUM149 and SUM159 cells 72 h after nucleofection with control or two independent siRNAs against CEBPD. h qRT-PCR analysis of CXCL8 and CEBPD mRNA in SUM159 xenograft tumors with Dox-inducible shRNA and with and without Dox-treatment as described [18]. Quantitative data are mean±S.E.M; a–c, g, n = 3; e, n = 4; h, n = 6; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant.

Fig 5: Human neutrophils migrate toward recombinant chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand (CCL)2 and CCL7. Human neutrophils were isolated from the blood of healthy volunteers and chemotaxis was measured across 3 µm membranes (ChemoTX, NeuroProbe) in response to recombinant human CXCL8, CCL2 and CCL7 (A), over a range of concentrations as stated. Neutrophil chemotaxis was also measured in response to a suboptimal concentration of recombinant human CXCL8 (5 ng/mL) in combination with recombinant human CCL2 (B) or recombinant human CCL7 (C) across a range of concentrations as stated (n=3 independent experiments using different healthy volunteer donors). Neutrophil chemotaxis was measured as the chemotactic index (number of migrated neutrophils following treatment vs medium control) after 1 hour. Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.