Journal: Frontiers in Immunology

Article Title: Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

doi: 10.3389/fimmu.2023.1142492

Figure Lengend Snippet: Uptake of zymosan by human intestinal epithelial cells. (A) SW480 cells were seeded on glass-bottom chambers as indicated in Methods, and fed overnight with pHrodo-red zymosan (zymosan, red) and counter stained with Hoechst 33342 (blue) prior to confocal live imaging. White arrowheads – intracellular red fluorescent zymosan, Black arrowheads - extracellular intact zymosan, arrow- intracellular fragmented zymosan. Original magnification x20, scale bar 10 µm. (B–E) Zymosan uptake is sensitive to cytochalasin-D. (B, C) SW480 were treated as in A, in the absence (B) or presence (C) of cytochalasin-D (CytoD, 10 µM). Scale bar 20 µm. Arrows and arrowhead indicate intracellular processed and intact zymosan respectively. Wider fields of the images are shown is Supplementary Figure S3 . (D) Phagocytosis was quantified using imageJ as the percentage of red-fluorescence positive cells in at least 4 randomly taken fields as described in Methods. Each dot is the quantification of a single field. Data is representative of three independent experiments performed. ***p ≤ 0.001, Unpaired t-test vs. no inhibitor. (E) SW480 cells were seeded in 96 well plate, treated as in (B, C) as well as with the vehicle (DMSO, 1:1000) in triplicate wells, and phagocytosis was assessed as the relative fluorescence (RFU) by a microplate reader. Data are shown as the individual measure of each biological replica and mean ± SD of biological triplicates from a representative of three independent experiments performed. ns-non significant ****p<0.0001, One-way ANOVA followed by Tukey multiple comparison test. (F–I) Zymosan uptake depends on Dectin-1. (F, G) SW480 were treated as in A, in the absence (F) or presence (G) of laminarin (1 mg/ml) that was added to the medium 1 hour prior to zymosan. Scale bar 50 µm. White arrows and arrowhead indicate intracellular processed and intact zymosan respectively. Black arrowheads indicate extracellular zymosan. (H) Phagocytosis was quantified as in (D) . (I) cells were seeded on 96 wells, treated as in (F, G) in triplicate wells, and phagocytosis was analyzed as in (E) . (J) Zymosan phagocytosis is resistant to Syk inhibition. SW480 cells were seeded on 96 well plate, in the presence or absence of the Syk inhibitor 574711 (1 and 5 µM), which was added 1 hour prior to the addition of pHrodo-red zymosan. Phagocytosis was assessed as in (E) . Data are shown as individual measures and mean ± SD of biological triplicates from a representative of three independent experiments performed. (K) Zymosan-induced IL-8 secretion is sensitive to Syk inhibitor. Cells seeded on the same 96 well plate were pre-treated with Syk inhibitor as in (J) and stimulated overnight with 100 μg/ml of non-labelled zymosan. Supernatants were assessed for IL-8 by ELISA. Data are shown as individual measures and mean ± SD of biological duplicates from a representative of three independent experiments performed. N.D- not detected; ns-non significant *p<0.05; **p<0.01, One-way ANOVA followed by Tukey multiple comparison test.

Article Snippet: Human colon epithelial cell lines SW480, HCT116 and Caco-2 were purchased from ATCC (Manassas, VA).

Techniques: Staining, Imaging, Fluorescence, Comparison, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

doi: 10.3389/fimmu.2023.1142492

Figure Lengend Snippet: LC3 is recruited to phagosomes in IECs. (A) SW480 LC3-GFP cells were fed with pHrodo-red zymosan overnight. Live imaging shows LAPosomes (arrow) as LC3 (green) around intact zymosan (red) particles, as well as fragmented zymosan and autophagosomes. (B) Fluorescence intensity profile along the arrow of an inset from (A) is shown on the graph. (C) Colonic organoids were fed with pHrodo-red zymosan (red) overnight and stained with LC3 antibody (green) and DAPI (blue). (D–F) Colonic organoids were fed with Rhodamine-green-X HK- C. albicans (green) overnight and stained with LC3 antibody (magenta) and DAPI (blue). Shown is LAP of yeast (D) and hyphal form (E, F) of HK- C. albicans . F is an inset of (E) Original magnification ×40 (A, B) , ×63 (C, D) x20 (E, F) scale bar 10 µm (A–D, F) and 50 µm (E) .

Article Snippet: Human colon epithelial cell lines SW480, HCT116 and Caco-2 were purchased from ATCC (Manassas, VA).

Techniques: Imaging, Fluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

doi: 10.3389/fimmu.2023.1142492

Figure Lengend Snippet: DPI inhibits zymosan phagocytosis. (A) Colonic organoid monolayers grown in differentiation medium for 3 days were exposed to pHrodo-red zymosan for 24 hours in the presence or absence of DPI (2 µM) that was added for 60 minutes prior to the addition of zymosan. Shown are intracellular fluorescent zymosan (red) and the organoid cells (DIC) of representative frames from 7-15 random fields imaged. The experiment was repeated 3 times using organoids from two different individuals. Original magnification x20, scale bar 50 µm. (B) Colonic organoids were seeded in 96 well plate, treated as in (A) in the presence or absence of DPI (2 or 10 µM) or in the presence of vehicle (DMSO 1:1000) in 8-replicate wells for 48 hours. Phagocytosis was assessed as the relative fluorescence by a microplate reader. (C) A summary of four experiments performed showing zymosan (zym) phagocytosis inhibition by 2 µM DPI in colonic organoids from two individuals (colon-2 and colon-3). (D) SW480 cells were seeded in 96 well plate and treated as in (B) in triplicates for 24 hours. Data are shown as individual measures (dots) and the mean ± SD of biological 8-replicates (B) and triplicates (D) from a representative of four or three independent experiments performed. **p<0.01, ***p<0.001, ****p<0.0001, One-way ANOVA followed by Tukey multiple comparison test (B, D) or individual t-test vs. no inhibitor for each of the separate experiments shown in (C) .

Article Snippet: Human colon epithelial cell lines SW480, HCT116 and Caco-2 were purchased from ATCC (Manassas, VA).

Techniques: Fluorescence, Inhibition, Comparison

Journal: Frontiers in Immunology

Article Title: Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

doi: 10.3389/fimmu.2023.1142492

Figure Lengend Snippet: Phagocytosed particles are directed to lysosomal processing. (A) Colonic organoids were incubated overnight with Rhodamine-Green-X labeled HK- C. albicans (green) and stained with lysosomal-NIR reagent (magenta). (B, C) Ileal organoids (B) and SW480 cells (C) were incubated with pHrodo-red zymosan (red) and stained with lysosomal-green reagent. Arrows indicate colocalization of fragmented HK- C. albicans or zymosan and lysosomes. (D) SW480 cells were fed with HK- C. albicans (green) and stained with LAMP2 antibody. (E) Intact and fragmented zymosan particles are surrounded by LAMP2. Ileal organoids were fed with AF488-zymosan (green) overnight, and stained with LAMP2 antibody (red). Arrowhead - intact zymosan, arrow- fragmented zymosan. (F, G) LAPosomes merge with lysosomes. (F) SW480 cells were fed with pHrodo red zymosan (red) and stained with LAMP2 (green) and LC3 (magenta) antibodies and counterstained with DAPI (blue). (G) Ileal organoids were fed with AF488-zymosan (green) and stained with LAMP2 (red) and LC3 (magenta) antibodies and counterstained with DAPI (blue). Original magnification x63 (A, D–G) , x40 (B) , x20 (C) , scale bar 10 µm.

Article Snippet: Human colon epithelial cell lines SW480, HCT116 and Caco-2 were purchased from ATCC (Manassas, VA).

Techniques: Incubation, Labeling, Staining

Journal: Cancers

Article Title: Role of N-Cadherin in Epithelial-to-Mesenchymal Transition and Chemosensitivity of Colon Carcinoma Cells.

doi: 10.3390/cancers14205146

Figure Lengend Snippet: Figure 1. Comparison of miRNA, mRNA and protein levels of selected epithelial to mesenchymal transition (EMT) and cell migration potential markers as measured by RT-PCR and Western blot analysis (Materials and methods). (A) mRNA and miRNA of EMT markers in HCT8 and HCT8_N- cad cells; (B) mRNA and miRNA of EMT markers in SW480 and SW480_N-cad cells; (C) mRNA and miRNA of EMT markers in 47B and 48 primocultures; (D) protein expression of selected EMT and cell migration potential markers in HCT8, SW480 and its _N-cad variants, 47B and 48B primocultures. Numbers indicate densitometric analysis results. B2M was used as a reference gene in RT-PCR. * p < 0.05 N-cadherin plasmid-treated vs. untreated cells, resp. 47B vs. 48B cells. α-tubulin was used as a reference protein in immunoblotting. * p < 0.05 plasmid N-cadherin-treated vs. untreated cells, resp. 47B vs. 48B; # p < 0.05 47B vs. 48B. Original western blots can be found in Supplementary File S1.

Article Snippet: Human epithelial adenocarcinoma colon cancer cell lines SW480 and HCT8 were purchased from ATCC (LGC Standards, Poland).

Techniques: Comparison, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Plasmid Preparation