Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model

doi: 10.3389/fimmu.2021.781185

Figure Lengend Snippet: IDO1 inhibitor mixed with MSCs improved the efficacy of MSCs to promote cartilage regeneration in OA animal model. (A) The levels of IL-1β in the OA animal model after one month of model establishment compared to normal control. (B) The levels of TNF-α in the OA animal model after one month of model establishment compared to normal control. (C) The levels of IFN-γ in the OA animal model after one month of model establishment compared to normal control. (D) The schematic diagram presented the performed strategy for stimulated MSCs to be used in the cartilage repair for the OA model. (E) HE staining for knee joints of OA animal model after one month that presented the initiation of cartilage degeneration compared to normal control groups. (F) Safranin-O staining for the cartilage of OA model after two months of treatment with Epacadostat-treated MSCs, IDO1-treated cells, without treatment group, normal control group. *** p < 0.001 vs NC group.

Article Snippet: The performance of IDO1-siRNA was validated by western blot using anti-human anti-IDO1 (Sinobiological, China) compared to anti-GAPDH.

Techniques: Animal Model, Staining

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model

doi: 10.3389/fimmu.2021.781185

Figure Lengend Snippet: IDO1 decreases the expression of chondrogenic genes in the stimulated MSCs. (A) Flow cytometry surface epitope profiling of MSCs. (B) The effect of IDO1 compared to Epacadostat on the chondrogenic differentiation potency of MSCs. (C) The expression of Sox9 under the effect of IDO1 compared to Epacadostat. (D) The expression of COL2A1 under the effect of IDO1 compared to Epacadostat. (E) The expression of the Ihh gene under the effect of IDO1 compared to Epacadostat. (F) The expression of Aggrecan under the effect of IDO1 compared to Epacadostat. (G) The expression of COL2A1 under the effect Epacadostat using IF assay. (H) Epacadostat promotes Sox9 signaling compared to IDO1. *** p < 0.001 and ** p < 0.01 vs NC group.

Article Snippet: The performance of IDO1-siRNA was validated by western blot using anti-human anti-IDO1 (Sinobiological, China) compared to anti-GAPDH.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model

doi: 10.3389/fimmu.2021.781185

Figure Lengend Snippet: IDO1 regulates the expression of Sox9 through activating β-catenin in the MSCs. (A) Silencing of IDO1 using siRNA in the MSCs. (B) The immunofluorescence expression of Sox9 and β-catenin in the IDO1 silenced cells compared to IDO1-positive cells. (C) The expression of Sox9, COL2A1, and β-catenin by western blot in the IDO1 knocked cells compared to active IDO1. (D) The knocking down of β-catenin in the SF-MSCs using siRNA transfection. (E) The expression of Sox9 under β-catenin silencing in the SF-MSCs compared to active β-catenin using flow cytometry. (F) The expression of Sox9 under β-catenin silencing using confocal laser microscopy. (G) The expression of Sox9 under β-catenin silencing by qPCR. (H) The expression of GSK3β in the MSCs under the effect of IDO1 and Epacadostat compared to the NC group. (I) The expression of GSK3α in the MSCs under the effect of IDO1 and Epacadostat compared to the NC group. (J) The expression of APC in the MSCs under the effect of IDO1 and Epacadostat compared to the NC group. ** p < 0.01 vs NC group. ns, no significance.

Article Snippet: The performance of IDO1-siRNA was validated by western blot using anti-human anti-IDO1 (Sinobiological, China) compared to anti-GAPDH.

Techniques: Expressing, Immunofluorescence, Western Blot, Transfection, Flow Cytometry, Microscopy

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model

doi: 10.3389/fimmu.2021.781185

Figure Lengend Snippet: IDO1 decreases the expression of GSK3β in MSCs through inducing its phosphorylation. (A) The immunofluorescence expression of pGSK3β showed increased expression under IDO1 effects, while it was reduced by Epacadostat, suggesting the inhibition of GSK3β phosphorylation by Epacadostat. (B) The western blot expression of pGSK3β under the effect of IDO1 compared to Epacadostat in the MSCs; the effect of two different concentrations showed a change in the expression of phosphorylation protein. (C) The expression of pGSK3β under the effect of IDO1 compared to Epacadostat by flow cytometry. (D) The expression of Wnt1 in the MSCs under the effect of Epacadostat compared to IDO1. (E) The immunofluorescent expression of Wnt1 in the MSCs under the effect of Epacadostat compared to IDO1. (F) The expression of the AhR receptor and β-catenin in the MSCs under the effect of Epacadostat compared to IDO1. *** p < 0.001, and ** p < 0.01.

Article Snippet: The performance of IDO1-siRNA was validated by western blot using anti-human anti-IDO1 (Sinobiological, China) compared to anti-GAPDH.

Techniques: Expressing, Immunofluorescence, Inhibition, Western Blot, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model

doi: 10.3389/fimmu.2021.781185

Figure Lengend Snippet: IDO1 induces the anti-localization interaction between β-catenin and Sox9 in the MSCs. (A, B) Bioinformatic analysis of P-P interaction between β-catenin and Sox9 using the database of String-db.org and thebiogrid.org. (C) Colocalization assay between β-catenin and Sox9 using immunofluorescence overlapping. (D) The analysis of expression intensity. (E) The calculation of intensity by frequency of every protein expression. (F) The normalizing intensity by calculating Pearson correlation. ** p < 0.01 Epacadostat vs IDO1.

Article Snippet: The performance of IDO1-siRNA was validated by western blot using anti-human anti-IDO1 (Sinobiological, China) compared to anti-GAPDH.

Techniques: Immunofluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model

doi: 10.3389/fimmu.2021.781185

Figure Lengend Snippet: IDO1 impairs chondrogenic differentiation of MSCs by activating Wnt1/β-catenin through downregulation of miR-122-5p. (A, B) The density and distribution of miRNA in the total RNA obtained from MSCs-treated IDO1 compared to normal. (C) The screening of up-/downregulated miRNA, which shows the downregulation of miR-122-5p under the effect of IDO1. (D) The levels of miR-122-5p in the MSCs treated with Epacadostat compared to IDO1 and NC cells. (E) The target sequence of miR-122-5p in the sequence of the Wnt1 gene. (F) The expression of Wnt1 protein under miR-122-5p mimics compared to miR-122-5p inhibitor. (G) qPCR confirmative experiment for Wnt1 expression under miR-122-5p mimics compared to inhibitor in the MSCs. *** p < 0.001, and ** p < 0.01 vs inhibitor group.

Article Snippet: The performance of IDO1-siRNA was validated by western blot using anti-human anti-IDO1 (Sinobiological, China) compared to anti-GAPDH.

Techniques: Sequencing, 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