Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

doi: 10.1016/j.biopha.2022.113672

Figure Lengend Snippet: Fig. 1. Propionate-mediated cytotoxicity and MUC2 production in LS 174 T cells. LS 174 T cells were treated with various concentrations of propionate at 37 ◦C for 24 h, and cytotoxicity was measured by MTT assay (A). Expression of MUC2 (B) was measured by quantitative real-time PCR. Total and intracellular MUC2 protein were detected by ELISA (C) and immunofluorescence (D) and quantified (E), respectively. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. Student’s t-test was used to determine the significance of the differences.

Article Snippet: The Human MUC2 ELISA kit (E-EL-H0632, Elabscience, Houston, TX, USA) was used to measure total MUC2 levels.

Techniques: MTT Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

doi: 10.1016/j.biopha.2022.113672

Figure Lengend Snippet: Fig. 3. Hypoxic response of LS 174 T cells to propionate treatment. Cells were treated with propionate (1–4 mM) for 24 h, and changes in HIF expression were investigated. (A) The transcriptional level of the α subunits of HIFs at 24 h. (B) The results of a time-course assay comparing MUC2 levels with those of the HIFs using qPCR. (C and D) Cell lysates were fractionated into cytosolic and nuclear fractions, and activated HIF-α levels were detected by western blot analysis (C) and quantified (D). Data are expressed as the mean ± SD of three independent experiments performed in triplicate. Student’s t-test was used to determine the significance of the differences: ns, not significant. NC, negative control; Pro, propionate-treated. The full-length blots of (C) are shown in Fig. S4.

Article Snippet: The Human MUC2 ELISA kit (E-EL-H0632, Elabscience, Houston, TX, USA) was used to measure total MUC2 levels.

Techniques: Expressing, Western Blot, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

doi: 10.1016/j.biopha.2022.113672

Figure Lengend Snippet: Fig. 4. HIF-2α was activated by propionate-induced mitochondrial respiration. (A) Propionate (4 mM) and oligomycin A (1 μM) were added to LS 174 T cells and incubated for 12 h, and ATPase activity was measured. (B–D) The suppressive effects of oligomycin A on HIF-α were investigated at the transcriptional level (B) and nuclear protein levels were measured by western blotting (C) and quantified (D). (E) Expression level of MUC2 was measured by qPCR. (F) Total MUC2 level was measured by ELISA. (G and H) Intracellular MUC2 (magnification 600 ×; scale bars: 50 µm) was measured immunofluorescence (G) and quantified (H). Linear correlation of MUC2 and HIF-2α expression by propionate (I). Data are expressed as the mean ± SD of three independent experiments performed in triplicate. One- way ANOVA followed by Tukey’s HSD was used to determine significance: ns, not significant. NC, negative control; Pro, propionate-treated. The full-length blots of (C) are shown in Fig. S5.

Article Snippet: The Human MUC2 ELISA kit (E-EL-H0632, Elabscience, Houston, TX, USA) was used to measure total MUC2 levels.

Techniques: Incubation, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

doi: 10.1016/j.biopha.2022.113672

Figure Lengend Snippet: Fig. 5. HIF-2α is the primary regulator of MUC2 expression under hypoxia. LS 174 T cells were transfected with mock siRNA or HIF-2α siRNA. (A–C) The effect of 4 mM propionate on the HIF-α subunits was measured at the gene level (A) and nuclear protein levels of HIF-α subunits were measured by western blotting (B) and quantified (C). (D–G) The suppressive effect of HIF-2α siRNA on MUC2 expression was detected at the gene level (D), total protein level of MUC2 was measured by ELISA (E), and intracellular MUC2 protein level (magnification 600 ×; scale bars: 50 µm) was measured by immunofluorescence (F) and quantified (G). Data are expressed as the mean ± SD of three independent experiments performed in triplicate. One-way ANOVA followed by Tukey’s HSD was performed: ns, not significant. NC, negative control; Pro, propionate-treated. The full-length blots of (B) are shown in Fig. S6.

Article Snippet: The Human MUC2 ELISA kit (E-EL-H0632, Elabscience, Houston, TX, USA) was used to measure total MUC2 levels.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

doi: 10.1016/j.biopha.2022.113672

Figure Lengend Snippet: Fig. 7. Propionate metabolism via a β-oxidation-like pathway. LS 174 T cells were transfected with mock siRNA or ACADS siRNA. The cells were then treated with propionate (4 mM) for 24 h. (A) The expression level of ACADS was measured by qPCR. (B) ATPase activity. (C) Expression of the HIF genes was measured by qPCR. (D and E) Nuclear level of HIF proteins were analyzed by western blotting (D) and quantified (E). (F) Expression level of the MUC2 was measured by qPCR. (G) Total MUC2 protein level was measured by ELISA. (H and I) Intracellular MUC2 (magnification 600 ×; scale bars: 50 µm) was measured by immunofluorescence (H) and quantified (I). Data are expressed as the mean ± SD of three independent experiments performed in triplicate. One-way ANOVA followed by Tukey’s HSD was used to determine the significance of differences: ns, not significant. NC, negative control; Pro, propionate-treated; ACADS, short-chain specific acyl-CoA dehydrogenase. The full-length blots of (D) are shown in Fig. S7.

Article Snippet: The Human MUC2 ELISA kit (E-EL-H0632, Elabscience, Houston, TX, USA) was used to measure total MUC2 levels.

Techniques: Transfection, Expressing, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

doi: 10.1016/j.biopha.2022.113672

Figure Lengend Snippet: Fig. 8. Hypoxia induced by propionate is not relevant to the carboxylation pathway in LS 174 T cells. LS 174 T cells were transfected with mock-siRNA or MUT siRNA. The cells were then treated with propionate (4 mM) for 24 h. (A) Expression level of MUT was measured by qPCR. (B) ATPase activity. (C) Expression levels of the HIF genes were measured by qPCR. (D and E) The nuclear levels of HIF proteins were analyzed by western blotting (D) and quantified (E). (F) Expression level of the MUC2 gene was measured by qPCR. (G) Total MUC2 level was measured by ELISA. (H and I) Intracellular MUC2 levels were measured by immunofluorescence (magnification 600 ×; scale bars: 50 µm) (H) and quantified (I). Data are expressed as the mean ± SD of three independent experiments performed in triplicate. One- way ANOVA followed by Tukey’s HSD was used to determine the significance of the differences: ns, not significant. NC, negative control; Pro, propionate-treated; MUT, methylmalonyl-CoA mutase. The full-length blots of (D) are shown in Fig. S8.

Article Snippet: The Human MUC2 ELISA kit (E-EL-H0632, Elabscience, Houston, TX, USA) was used to measure total MUC2 levels.

Techniques: Transfection, Expressing, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Negative Control

Journal: Advanced functional materials

Article Title: A Pumpless, High-Throughput Microphysiological System to Mimic Enteric Innervation of Duodenal Epithelium and the Impact on Barrier Function

doi: 10.1002/adfm.202409718

Figure Lengend Snippet: Our MPS system enables traditional barrier strength measures like transepithelial electrical resistance (TEER) and apparent permeability assay. a) TEER was measured via chopstick electrodes and the EVOM2. b) TEER values were overall greater for cultures containing the epithelium (Epi); n = 3, m = 3; error bars = SD; ** = p < 0.01, one-way ANOVA with multiple comparisons. c) Fold change of the TEER values controlled for the 3D gel layer degrading when ENS was cultured in it; n = 3, m = 3; error bars = SD; * = p < 0.05; two-tailed unpaired t -test. d) Apparent permeability, measured by lucifer yellow diffusion through the culture, followed the TEER results. e) Less fluorophore traveled through the layers when there was an epithelium present (n = 3, m = 3; error bars = SD; ** = p < 0.01; one-way ANOVA with multiple comparisons) and f) the fold change comparison to appropriate controls showed a significant difference between the epithelium with and without the ENS (n = 3, m = 3; error bars = SD; * = p < 0.05, *** = p < 0.001; two-tailed unpaired t -test). g) Cell area coverage was also measured using phalloidin-stained monolayers and images of the entire culture area. h) The concentration of Muc2 is higher in the epithelium containing cultures, as well as the co-culture show a trending increase in Muc2 production compared to the epithelium alone (n = 3, m = 2–3; error bars = SD; * = p < 0.05; one-way ANOVA with multiple comparisons). i) A higher EGF basal supernatant concentration was recorded from the co-culture condition than either of the monoculture conditions (n = 3, m = 2–3; error bars = SD; * = p < 0.05; *** = p < 0.001; Kruskal–Wallis test for non-parametric data).

Article Snippet: A Muc2 ELISA ( Novus Biologicals , NBP276701) was performed similarly using the apical supernatants.

Techniques: Permeability, Cell Culture, Two Tailed Test, Diffusion-based Assay, Comparison, Staining, Concentration Assay, Co-Culture Assay

Journal: Scientific reports

Article Title: Robust and reproducible human intestinal organoid-derived monolayer model for analyzing drug absorption.

doi: 10.1038/s41598-025-95823-z

Figure Lengend Snippet: Fig. 6. Characterization of the novel human duodenal organoid-derived monolayer model. (a) Immunofluorescence analysis for E-cadherin (red), ZO-1 (green), (b) MUC2 (red), and nucleus (blue). Scale bars, 100 µm. (c) Immunofluorescence analysis depicting cross sections of the monolayer model; ZO-1 (green), nucleus (blue), and F-actin (white). Scale bars, 100 µm. Black arrows indicate the Transwell membrane. (d) and (e) Transmission electron microscopic analysis of the monolayer model. Mv, microvilli; Tj, tight junction; Sg, secretory granule-like structures. Scale bars, 10 µm for low magnification images and 2 µm for high magnification images. (f) Efflux transporter P-gp assay using the P-gp substrate digoxin with or without zosuquidar as an inhibitor. AtoB, apical to basal; BtoA, basal to apical. (g) Efflux transporter assay using BCRP substrate sulfasalazine with or without Ko143. (h) UDP-glucuronosyltransferase (UGT) functional assay using raloxifene as a UGT substrate. (i) Carboxylesterase 2 (CES2) functional assay using irinotecan as a substrate with or without loperamide as an inhibitor. The amount of SN-38, a metabolite of irinotecan, was detected in the cells of the monolayer model. (j) and (k) Midazolam permeability and metabolism assay using 2 µM midazolam as a substrate of CYP3A with or without 1-aminobenzotriazole (ABT) as a pan-CYP inhibitor. BLQ, below limit of quantification. (l) Relative gene expression determined by quantitative RT-PCR of the monolayer model with induction of VD3 or VD3 + rifampicin. Cells were treated with inducers for the last two days of the differentiation process. The P value was determined by a two-tailed unpaired t-test.

Article Snippet: The monolayer was incubated with primary antibodies [anti-ZO-1 antibody Alexa Fluor 488 (MA3-39,100-A488; Thermo Fisher Scientific), anti-E-cadherin antibody (PA5-32,178; Thermo Fisher Scientific), and anti-MUC2 antibody (NBP225,221; Novus Biologicals)] in blocking solution containing 0.3% Triton X-100 at 4 °C overnight.

Techniques: Derivative Assay, Immunofluorescence, Membrane, Transmission Assay, Functional Assay, Permeability, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Enhanced Utilization of Induced Pluripotent Stem Cell–Derived Human Intestinal Organoids Using Microengineered Chips

doi: 10.1016/j.jcmgh.2017.12.008

Figure Lengend Snippet: Table of Antibodies Used in Study

Article Snippet: Mucin-2 (CCP58) , 1:500 , NBP2-25221 , Mouse IgG1 , Novus Biologicals.

Techniques: