Journal: Theranostics

Article Title: Exosomes-mediated Transfer of miR-125a/b in Cell-to-cell Communication: A Novel Mechanism of Genetic Exchange in the Intestinal Microenvironment

doi: 10.7150/thno.41802

Figure Lengend Snippet: Identification of MCL1 as a miR-125a/b target. (A) Schematic description of the hypothetical duplex formed by the interactions between the binding site in the MCL1 3'-UTR and miR-125a/b. The miR-125a/b seed sequence and the seed sequence binding sites in the MCL1 3'-UTR are indicated in red. All nucleotides of the seed sequence of the binding site are conserved in several species, including human, mouse, rat and rabbit. The calculated free energy values of the hybrids are indicated. (B) qPCR analysis of MCL1 mRNA levels in HIEC6 cells transfected with miR-125a/b mimic, inhibitor, and scramble negative control. (C-D) Representative blots and relative quantification of MCL1 protein levels in HIEC6 cells transfected with miR-125a/b mimic, inhibitor, and scramble negative control. (E-F) Representative blots and relative quantification of MCL1 protein levels in HIEC6 cells transfected with NC mimic, miR-125a mimic or biotinylated miR-125a mimic. (G) qPCR analysis of MCL1 and GAPDH mRNA levels in HIEC6 after pulling down with control probe or miR-125a probe. (H) The relative luciferase activities in HIEC6 transfected with wild type or mutant MCL1 3'-UTR. N=3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Mammalian MCL1 overexpression plasmids and an empty plasmid (control plasmid) were purchased from Genescript (China). siRNA designed to specifically silence MCL1 and scrambled siRNA (control siRNA) were purchased from RioboBio.

Techniques: Binding Assay, Sequencing, Transfection, Negative Control, Quantitative Proteomics, Control, Luciferase, Mutagenesis

Journal: Theranostics

Article Title: Exosomes-mediated Transfer of miR-125a/b in Cell-to-cell Communication: A Novel Mechanism of Genetic Exchange in the Intestinal Microenvironment

doi: 10.7150/thno.41802

Figure Lengend Snippet: MCL1 promoted proliferation and inhibited apoptosis of HIEC6 cells. (A) qPCR analysis of MCL1 mRNA levels in HIEC6 cells transfected with MCL1 plasmid, Control plasmid, MCL1 siRNA, or scrambled Control siRNA. (B-C) Representative blots and relative quantification of MCL1 protein in HIEC6 cells transfected with MCL1 plasmid, Control plasmid, MCL1 siRNA, or scrambled Control siRNA. (D) CCK-8 were performed 12, 24, 36, 48 and 60 h after the transfection of HIEC6 with MCL1 siRNA, Control siRNA, MCL1 plasmid, or Control plasmid. (E) Representative images of EdU staining in HIEC6 transfected with MCL1 plasmid, Control plasmid, MCL1 siRNA, or scrambled Control siRNA. (C) Quantitative assessment of percentage of EdU positive cells in (F). (G) Representative flow cytometry plots showing the percentages of early apoptotic cells (Annexin V + /PI - ), late apoptotic cells (Annexin V + /PI + ) and total (early+late) apoptotic cells in HIEC6 transfected with MCL1 plasmid, Control plasmid, MCL1 siRNA, or scrambled Control siRNA. (H) Pooled flow cytometry data from (G). (I) Representative blots of PCNA and Cleaved C3 protein levels in HIEC6 transfected with MCL1 siRNA, Control siRNA, MCL1 plasmid, or Control plasmid. N=3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Mammalian MCL1 overexpression plasmids and an empty plasmid (control plasmid) were purchased from Genescript (China). siRNA designed to specifically silence MCL1 and scrambled siRNA (control siRNA) were purchased from RioboBio.

Techniques: Transfection, Plasmid Preparation, Control, Quantitative Proteomics, CCK-8 Assay, Staining, Flow Cytometry

Journal: Theranostics

Article Title: Exosomes-mediated Transfer of miR-125a/b in Cell-to-cell Communication: A Novel Mechanism of Genetic Exchange in the Intestinal Microenvironment

doi: 10.7150/thno.41802

Figure Lengend Snippet: miR-125a inhibited HIEC6 cell proliferation and promoted apoptosis by targeting MCL1. (A-B) Pilot study to determine the dosage of MCL1 vector to rescue miR-125a mimic induced MCL1 suppression. (C) CCK-8 were performed 12, 24, 36, 48 and 60 h after the co-transfection of HIEC6 with NC mimic+control vector, miR-125a mimic+control vector, NC mimic +MCL1 vector, miR-125a mimic+MCL1 vector. (D) Representative images of EdU staining in HIEC6 cells transfected with NC mimic+control vector, miR-125a mimic+control vector, NC mimic +MCL1 vector, miR-125a mimic+MCL1 vector. (E) Quantitative analysis of percentage of EdU positive cells in (D). (F-G) Representative flow cytometry plots and relative quantification of the ratio of early apoptotic cells (Annexin V + /PI - ), late apoptotic cells (Annexin V + /PI + ) and total (early late) apoptotic cells in HIEC6 transfected with NC mimic+control vector, miR-125a mimic+control vector, NC mimic +MCL1 vector, miR-125a mimic+MCL1 vector. (H) Gene expression profiles of proliferation marker PCNA and Ki67 mRNA levels in HIEC6 transfected with NC mimic+control vector, miR-125a mimic+control vector, NC mimic +MCL1 vector, miR-125a mimic+MCL1 vector. (I) Gene expression profiles of anti-apoptotic Bcl-2 and pro-apoptotic Bax mRNA levels in HIEC6 cells transfected with NC mimic+control vector, miR-125a mimic+control vector, NC mimic +MCL1 vector, miR-125a mimic+MCL1 vector. N=3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Mammalian MCL1 overexpression plasmids and an empty plasmid (control plasmid) were purchased from Genescript (China). siRNA designed to specifically silence MCL1 and scrambled siRNA (control siRNA) were purchased from RioboBio.

Techniques: Plasmid Preparation, CCK-8 Assay, Cotransfection, Control, Staining, Transfection, Flow Cytometry, Quantitative Proteomics, Gene Expression, Marker

Journal: Theranostics

Article Title: Exosomes-mediated Transfer of miR-125a/b in Cell-to-cell Communication: A Novel Mechanism of Genetic Exchange in the Intestinal Microenvironment

doi: 10.7150/thno.41802

Figure Lengend Snippet: GLP-2 exerted its intestinotrophic effect by decreasing exosomal miR-125a/b and thus upregulating MCL1 expression in vivo . (A-B) Representative blots and relative quantification of MCL1 protein level in jejunum mucosa of SBS rats treated with GLP-2 or Saline. (C) qPCR analysis of MCL1 mRNA levels in jejunum mucosa of SBS rats treated with GLP-2 or Saline. (D) Representative images of H&E staining, Ki67 staining and TUNEL staining of remaining jejunum in Sham, SBS+NC loaded IMF-Exo and SBS+miR-125a loaded IMF-Exo rats. (E) Quantification of intestinal epithelial thickness in (D). (F) Quantitative analysis of percentage of Ki67 positive cells in (D). (G) Quantitative analysis of percentage of TUNEL positive cells in (D). N=5-8, **p < 0.01, ***p < 0.001.

Article Snippet: Mammalian MCL1 overexpression plasmids and an empty plasmid (control plasmid) were purchased from Genescript (China). siRNA designed to specifically silence MCL1 and scrambled siRNA (control siRNA) were purchased from RioboBio.

Techniques: Expressing, In Vivo, Quantitative Proteomics, Saline, Staining, TUNEL Assay