Fig 2: MUC5AC, Sp1 and miR-130b levels in an intrahepatic bile duct stone animal model. (A) Sprague-Dawley rats underwent laparotomy and a PE tube was inserted into their common bile duct and fixed. The PE tube was placed from the back of the neck through a subcutaneous tunnel and fixed. (B) Days of each drug injection. (C) Rat serum levels of AST, ALT and TB were measured. (D) Images of rat bile smears after modeling. (E) Western blotting was performed to measure Sp1 expression in bile duct tissues from different groups. (F) RT-qPCR assay to detect expression of miR-130b in bile duct tissues across different groups. (G) RT-qPCR assay to detect expression levels of Sp1 and MUC5AC in bile duct tissues from different groups. (H) Correlation analysis of Sp1 and miR-130b mRNA expression levels. (I) Correlation analysis of MUC5AC and Sp1 mRNA expression levels. *P<0.05, **P<0.01 and ***P<0.0001. AST, aspartate transaminase; ALT, alanine aminotransferase; TB, total bilirubin; MUC5AC, mucin 5AC; Sp1, specificity protein 1; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; PE, polyethylene.

Fig 3: Detection of miR-130b, Sp1 and MUC5AC expression in HIBEpiCs following LPS treatment. (A) ELISA was performed to detect changes in MUC5AC expression in HIBEpiC supernatant. (B) RT-qPCR was performed to detect changes in MUC5AC and Sp1 mRNA expression in HIBEpiCs. (C) Western blotting was performed to detect changes Sp1 protein expression in HIBEpiCs. (D) RT-qPCR was performed to detect changes in miR-130b expression in HIBEpiCs. (E) immunofluorescence experiments for HIBEpiCs were performed to detect the transmembrane location of Sp1 in the control group compared with the 10 µg/ml LPS treatment group. Magnification, ×40. *P<0.05, **P<0.01. MUC5AC, mucin 5AC; Sp1, specificity protein 1; miR, microRNA; HIBEpiCs, human intrahepatic biliary epithelial cells; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative PCR.

Fig 4: Detection of MUC5AC expression in normal bile duct tissue and in tissues from patients with hepatoliths. (A) Immunohistochemistry assay of MUC5AC staining in paraffin-embedded sections from normal human subjects and patients with hepatoliths at different magnifications and quantitative analysis of MUC5AC expression level of each section. (B) Co-staining immunofluorescence experiments for MUC5AC and Sp1 expression in sections from normal human subjects and patients with hepatoliths (magnification, ×400), with quantitative analysis of Sp1 expression levels in each section. (C) Reverse transcription-quantitative PCR experiments for miR-130b expression in clinical tissue from normal human subjects and patients with hepatoliths. n=8 in the control group; n=10 in the hepatolith group. **P<0.01. MUC5AC, mucin 5AC; Sp1, specificity protein 1; miR, micro130b RNA.

Fig 5: Identifying the direct binding position of Sp1 to the MUC5AC promoter sequence and further verification of the regulatory effect of Sp1 by transfection. (A) An online database was used to predict the possible binding positions for Sp1 to the MUC5AC promoter sequence. (B) Chromatin immunoprecipitation-qPCR experiments showing the Sp1 binding site on the MUC5AC promoter sequence and the alterations of Sp1 binding intensity between control and experimental groups (10 µg/ml LPS pretreatment for 24 h). HIBEpiCs were transfected with shRNA to overexpress Sp1 or shRNA to inhibit Sp1 or pretreated with 10 µg/ml mithramycin A (an Sp1 inhibitor). (C) RT-qPCR detection of Sp1 mRNA expression in HIBEpiCs. (D) Western blotting assay of Sp1 protein expression in HIBEpiCs. (E) RT-qPCR detection of MUC5AC mRNA expression in HIBEpiCs. (F) ELISA detection of MUC5AC expression in HIBEpiC supernatant. *P<0.05, **P<0.01 and ***P<0.0001. MUC5AC, mucin 5AC; Sp1, specificity protein 1; HIBEpiCs, human intrahepatic biliary epithelial cells; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative PCR; shRNA, short hairpin RNA; NC, negative control; MA, mithramycin A.