Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Aberrant α(1,2)-Fucosylation at Ocular Surface Ameliorates Dry Eye Disease

doi: 10.3390/ijms22157863

Figure Lengend Snippet: Effects of 2-D-gal on conjunctival goblet cell and meibomian gland of the eyelid. ( A ) Representative images of periodic acid–Schiff (PAS) staining of the inferior conjunctival fornix (sagittal section; scale bar: 100 μm) and quantification of PAS-stained goblet cell counts in BALB/c mice under desiccating stress with or without 2-D-gal treatment. ( B ) ELISA for conjunctival MUC5AC. ( C ) Representative transillumination meibography images. ( D ) Quantification of the meibomian gland area of the upper and lower eyelids. A circle depicts data (mean ± SD) from a single individual animal. ns: not significant.

Article Snippet: The protein was extracted from the conjunctiva as described above and assayed for the concentration of MUC5AC by using mouse MUC5AC ELISA kit (Novus Biologicals, Centennial, CO, USA).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Guishaozichuan granules can attenuate asthma in rats via the MUC5AC/EGFR signaling pathway

doi: 10.3389/fphar.2022.1011751

Figure Lengend Snippet: Effects of GSZC granules on lung MUC5AC (A) , EGFR (B) mRNA expression in rats suffering from asthma. Values are the mean ± SD, n = 6 for each group. ▲▲ p < .01 versus the normal group, ** p < .01 versus the model group.

Article Snippet: MUC5AC antibody was purchased from Boster Group (20180824; Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Guishaozichuan granules can attenuate asthma in rats via the MUC5AC/EGFR signaling pathway

doi: 10.3389/fphar.2022.1011751

Figure Lengend Snippet: Effects of GSZC granules on lung MUC5AC (A) , EGFR (B) protein expression in rats suffering from asthma. Values are the mean ± SD, n = 6 for each group. ▲▲ p < .01 versus the normal group, * p < .05, ** p < .01 versus the model group.

Article Snippet: MUC5AC antibody was purchased from Boster Group (20180824; Wuhan, China).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Molecular mechanism underlying miR-130b-Sp1 transcriptional regulation in LPS-induced upregulation of MUC5AC in the bile duct epithelium

doi: 10.3892/mmr.2020.11745

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 250 μl concentrated cell supernatant solution was obtained from the control, LPS exposure, Sp1 shRNA transfection and miR130b mimics intervention groups and assayed using the human MUC5AC ELISA assay kit (cat. no. CSB-E10109h; Cusabio Technology, LLC) to determine MU5AC concentration.

Techniques: Expressing, Immunohistochemistry, Staining, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: Molecular mechanism underlying miR-130b-Sp1 transcriptional regulation in LPS-induced upregulation of MUC5AC in the bile duct epithelium

doi: 10.3892/mmr.2020.11745

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 250 μl concentrated cell supernatant solution was obtained from the control, LPS exposure, Sp1 shRNA transfection and miR130b mimics intervention groups and assayed using the human MUC5AC ELISA assay kit (cat. no. CSB-E10109h; Cusabio Technology, LLC) to determine MU5AC concentration.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Molecular mechanism underlying miR-130b-Sp1 transcriptional regulation in LPS-induced upregulation of MUC5AC in the bile duct epithelium

doi: 10.3892/mmr.2020.11745

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 250 μl concentrated cell supernatant solution was obtained from the control, LPS exposure, Sp1 shRNA transfection and miR130b mimics intervention groups and assayed using the human MUC5AC ELISA assay kit (cat. no. CSB-E10109h; Cusabio Technology, LLC) to determine MU5AC concentration.

Techniques: Animal Model, Injection, Western Blot, Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Molecular mechanism underlying miR-130b-Sp1 transcriptional regulation in LPS-induced upregulation of MUC5AC in the bile duct epithelium

doi: 10.3892/mmr.2020.11745

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 250 μl concentrated cell supernatant solution was obtained from the control, LPS exposure, Sp1 shRNA transfection and miR130b mimics intervention groups and assayed using the human MUC5AC ELISA assay kit (cat. no. CSB-E10109h; Cusabio Technology, LLC) to determine MU5AC concentration.

Techniques: Binding Assay, Sequencing, Transfection, Chromatin Immunoprecipitation, Control, shRNA, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Molecular Medicine Reports

Article Title: Molecular mechanism underlying miR-130b-Sp1 transcriptional regulation in LPS-induced upregulation of MUC5AC in the bile duct epithelium

doi: 10.3892/mmr.2020.11745

Figure Lengend Snippet: Investigation of the direct binding position between miR-130b and the 3′-UTR sequence of Sp1 and further verification of the regulatory effect of miR-130b on MUC5AC and Sp1 expression. (A) Online tools were used to predict the binding position of miR-130b to the 3′-UTR sequence of Sp1, and the corresponding luciferase primer for a natural plasmid and a mutated plasmid were established. (B and C) Luciferase gene detection for miR-130b and the Sp1 3′-UTR sequence. HIBEpiCs were transfected with miR-130b overexpression mimics or miR-130b inhibitors for 24 h. (D) RT-qPCR detected changes in miR-130b expression in HIBEpiCs. (E) RT-qPCR detected changes in of Sp1 mRNA expression in HIBEpiCs. (F) Western blotting detected changes Sp1 protein expression in HIBEpiCs. (G) RT-qPCR detected changes in of MUC5AC mRNA expression in HIBEpiCs. (H) ELISA detected changes in MUC5AC levels in HIBEpiC supernatant. *P<0.05, **P<0.01 and ***P<0.0001. MUC5AC, mucin 5AC; Sp1, specificity protein 1; miR, microRNA; HIBEpiCs, human intrahepatic biliary epithelial cells; RT-qPCR, reverse transcription-quantitative PCR; 3′-UTR, 3′-untranslated region; LPS, lipopolysaccharide; NC, negative control; mut, mutant; shRNA, short hairpin RNA; inb, inhibitor.

Article Snippet: Subsequently, 250 μl concentrated cell supernatant solution was obtained from the control, LPS exposure, Sp1 shRNA transfection and miR130b mimics intervention groups and assayed using the human MUC5AC ELISA assay kit (cat. no. CSB-E10109h; Cusabio Technology, LLC) to determine MU5AC concentration.

Techniques: Binding Assay, Sequencing, Expressing, Luciferase, Plasmid Preparation, Transfection, Over Expression, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Mutagenesis, shRNA

Journal: eBioMedicine

Article Title: Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids

doi: 10.1016/j.ebiom.2022.104132

Figure Lengend Snippet: Characterizing the cell types of undifferentiated hAOs and the permission of hAOs to 229E and OC43 infections. (A) to (D) Characterizing cell types by immunostaining Kertain 5 (basal cells), MUC5AC (goblet cells), β-tubulin (ciliated cells) and CC10 (club cells). (E) schematic diagram for seasonal coronaviruses inoculation in hAOs. (F) Dynamics of intracellular virus RNA copies in hAOs cultured at 33 °C from 1 to 24 h post-inoculation ( n =3). (G) Dynamics of infectious viral titers in hAOs cultured at 33 °C from 1 to 24 h post-inoculation ( n =3). (H) Immunofluorescence staining for viral double-stranded RNA (dsRNA) in hAOs at 33 °C. Scale bar = 25 μm. (I) Transmission electron microscopy analysis of 229E and OC43 infected hAOs cultured with expansion medium at 33 °C.

Article Snippet: Primary antibodies used in this study are as follows: anti-EpCAM antibody (1:1000, rabbit mAb; Abcam), anti-dsRNA antibody (1:500, mouse mAb; Scicons J2), anti-β-Tubulin antibody (1:500, rabbit mAb; Cell signaling), anti-MUC5AC antibody (1:500, rabbit mAb; Cell signaling), anti-Keratin 5 antibody (1:1000, rabbit mAb; Cell signaling), and anti-CC10 antibody (1:200, mouse mAb, Santa Cruz Biotechnology), human IVIg (1:50, Biotest).

Techniques: Immunostaining, Virus, Cell Culture, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Infection

Journal: eBioMedicine

Article Title: Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids

doi: 10.1016/j.ebiom.2022.104132

Figure Lengend Snippet: Recapitulating 229E and OC43 infections in differentiated hAOs. (A) Transcriptional levels of specific cell-type markers upon differentiation at 37 °C. (B) to (E) Characterizing cell types by immunostaining β-tubulin (ciliated cells), MUC5AC (goblet cells), Kertain 5 (basal cells) and CC10 (club cells) (Scale bar = 25 μm). (F) Dynamics of intracellular virus RNA copies in differentiated hAOs at 33 °C from 1 to 24 h post-inoculation ( n =3). (G) Dynamics of infectious viral titers in differentiated hAOs at 33 °C from 1 to 24 h post-inoculation ( n =3). (H) Immunofluorescence staining for viral double-stranded RNA (dsRNA) in differentiated organoids at 33 °C (Scale bar = 10 μm).

Article Snippet: Primary antibodies used in this study are as follows: anti-EpCAM antibody (1:1000, rabbit mAb; Abcam), anti-dsRNA antibody (1:500, mouse mAb; Scicons J2), anti-β-Tubulin antibody (1:500, rabbit mAb; Cell signaling), anti-MUC5AC antibody (1:500, rabbit mAb; Cell signaling), anti-Keratin 5 antibody (1:1000, rabbit mAb; Cell signaling), and anti-CC10 antibody (1:200, mouse mAb, Santa Cruz Biotechnology), human IVIg (1:50, Biotest).

Techniques: Immunostaining, Virus, Immunofluorescence, Staining