Journal: Gut microbes

Article Title: Detecting host responses to microbial stimulation using primary epithelial organoids.

doi: 10.1080/19490976.2023.2281012

Figure Lengend Snippet: Figure 3. Development and characterization of a 2D model for primary intestinal epithelial cells. (a-c) Detection of tight junction marker, ZO1 (red), F-actin with phalloidin (green), and nuclei, DAPI (blue) (a), enterocyte marker, ALDOB (red), and nuclei, DAPI (blue) (b) and goblet cell marker, MUC13 (red), and nuclei, DAPI (blue) (c) in confluent layers of intestinal epithelial cells. Scale bar 25 µm. Insert in (a) shows a Z-section of the cell layer with clear localization of ZO1 and F-actin at the apical surface. Scale bar 10 µm. (d) Left: Primary epithelial cells seeded in 2D form a confluent layer within 7 days from seeding. Right: MA-plot based on RNA-seq analysis of 2D vs 3D cells. Y axis shows 2D vs 3D log2FC, and X shows baseline expression in TPM (transcript per million). Color shows the number of genes in each bin. Full list of GO-terms enriched in 2D and 3D cultures in Table S2. (e) Gene set enrichment analysis of the uniquely annotated genes associated with 2D or 3D cultures versus published gene signatures representing enterocyte differentiation, general differentiation in the intestine, proliferation, and stem cells. X axis show observed vs expected overlap based on randomly selected genes. (f,g) UMAP plots of single-cell RNAseq data acquired using the 10× platform. c: Colors show cells cultured in 2D and 3D. d: Colors show nine cell clusters, defined by the Leiden method. (h) Cell distribution between the different clusters from panel j. Bars

Article Snippet: The following primary antibodies were used: anti-ZO1 (61-7300; Invitrogen), anti Muc13 (HPA045163; Atlas Antibodies), and anti-AldoB (HPA073201; Atlas Antibodies) and secondary antibodies Alexa Fluor 647 polyclonal donkey anti-rabbit.

Techniques: Marker, RNA Sequencing, Expressing, Cell Culture

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: (A) Identification of a putative miR-145-binding site in the MUC13 3′ UTR region. Seven bases (597 through 603) of the MUC13 3′ UTR are perfect matches (seed sequence) for miR-145 binding. (B) Comparison of the MUC13-binding element among mammals demonstrates a high degree of conservation. (C) MUC13 expression on miR-145 transfection was examined at protein and mRNA levels by Western blot analyses and semi-quantitative reverse transcription–PCR (RT-PCR), respectively. (D) Luciferase reporter assay was used to examine the miR-145-mediated regulation of gene expression. HPAF-II cells were transiently co-transfected for 48 h with reporter plasmids (0.5 μg, WT or MUT) and 100 nM of miR-145 or NC mimic using Lipofectamine 2000. Luciferase (Firefly; test and Renilla, transfection efficiency control) activity was assessed using a dual-luciferase assay system. Data are presented as normalized fold change in luciferase activity (mean ± SD; n= 3, * P <0.05).

Article Snippet: The slides were stained with anti-MUC13 MAb and HER2 using Biocare's MACH4 Universal HRP-Polymer kit (Biocare Medical, CA, USA).

Techniques: Binding Assay, Sequencing, Comparison, Expressing, Transfection, Western Blot, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Gene Expression, Control, Activity Assay

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: (A and B) Cells were transfected with miR-145 mimic, NC or miR-145 inhibitor in addition to miR-145 mimic for 48 h. Immunoblotting was performed for analysis of indicated proteins. (C and D) Confocal microscopy of HPAF-II, Capan-1 and HPAF-II sh-MUC13 cells treated with NC and miR-145 mimic. Data show a decrease in MUC13 (green) and HER2 (green) and an increase in p53 (pink) levels that reciprocated the results from the RNAi experiments using HPAF-II sh-MUC13 cells.

Article Snippet: The slides were stained with anti-MUC13 MAb and HER2 using Biocare's MACH4 Universal HRP-Polymer kit (Biocare Medical, CA, USA).

Techniques: Transfection, Western Blot, Confocal Microscopy

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: AsPC-1 cells (gemcitabine resistant cells) were transfected with miR-145 mimic or NC and then treated with a gemcitabine-conditioned medium (100 nM) for 48 h followed by the (A) matrigel invasion assay. Cells were photographed and counted using an imaging system. Bars represent mean ± SD; (n=3); *p<0.01 and **p<0.001. (B) Western blotting for the analysis of expression of HER2, MUC13 and the gemcitabine target, Mcl-1. (C) Flow cytometry analysis of miR-145-transfected human pancreatic ASPC-1 and HPAF-II cells indicating an increase in the G0–G1 stage. Data are representative of one of three similar experiments.

Article Snippet: The slides were stained with anti-MUC13 MAb and HER2 using Biocare's MACH4 Universal HRP-Polymer kit (Biocare Medical, CA, USA).

Techniques: Transfection, Invasion Assay, Imaging, Western Blot, Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: The antitumor effect of miR-145 was confirmed by in vivo experiments using xenograft models. (A) The antitumor effect of miR-145 was analyzed after intratumoral administration of miR-145 in established HPAF-II tumors. Average tumor volumes were calculated. Bars represent mean ± SD; *p<0.05 and **p<0.01. (B) Also, the xenograft tumors from miR-145 treated mice were analyzed for changes in MUC13 and HER2 expression (Original magnifications 40X) through IHC and miR-145 levels (Original magnifications 10X) using in situ hybridization followed by microscopy.

Article Snippet: The slides were stained with anti-MUC13 MAb and HER2 using Biocare's MACH4 Universal HRP-Polymer kit (Biocare Medical, CA, USA).

Techniques: In Vivo, Expressing, In Situ Hybridization, Microscopy

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: Immunohistochemistry and in situ hybridization was used to detect MUC13 and miR-145, respectively, on the tissue microarray slides (procured from US Biomax, Inc., Rockville, MD) in various (A) PanIN lesions (original magnifications: MUC13 60X; miR-145 20X), (B) adenocarcinoma (original magnifications 60X) and (C) adjacent normal (Adj) (original magnifications: MUC13 40X; miR-145 20X) and normal pancreatic cancer cells (original magnifications: 20X).

Article Snippet: The slides were stained with anti-MUC13 MAb and HER2 using Biocare's MACH4 Universal HRP-Polymer kit (Biocare Medical, CA, USA).

Techniques: Immunohistochemistry, In Situ Hybridization, Microarray