Journal: bioRxiv

Article Title: Effects of Alternative Splicing-Specific Knockdown of Tjp1 α+ by Rbm47 on Tight Junctions Assembly during Blastocyst Development

doi: 10.1101/2023.07.18.549609

Figure Lengend Snippet: (A) The position of both isoforms of Tjp1 (exon11/12; α+/α-), the α-domain (exon20/21) and two Tjp1 α-specific (exon 19/21; w/o α and alternatively spliced exon 27/28; α-) primers used for cDNA amplification of Tjp1 isoforms. Arrows mark the position and direction of primers used for cDNA synthesis. Transcription levels of Tjp1 α+/-, Tjp1 α variant (B), and Tjp1 α+/-, Tjp1 α-, Rbm47 (C). Transcription levels of Tjp1 α+/α-, Tjp1 α-, and Rbm47, shown as relative quantification (RQ). Error bars represent mean ± standard error.

Article Snippet: Primary antibodies against RBM47 (HPA006347, Sigma-Aldrich), TJP1 α+ (HP9044; Hycult Biotechnology, Uden, Netherlands) and TJP1 α-(HP9045; Hycult Biotechnology) were diluted and incubated with the embryos overnight at 4°C.

Techniques: Amplification, Variant Assay

Journal: bioRxiv

Article Title: Effects of Alternative Splicing-Specific Knockdown of Tjp1 α+ by Rbm47 on Tight Junctions Assembly during Blastocyst Development

doi: 10.1101/2023.07.18.549609

Figure Lengend Snippet: (A) Expression and localisation of Tjp1 α+ and Tjp1 α-in the Rbm47 KD blastocyst. (B) Gene expression patterns of Tjp1 variants from 8-cell to hatching blastocyst (C) Proximity ligation assay (PLA) showing the interaction between Tjp1(mainly Tjp1 α-) and Ocln in blastocysts. 8C(8-cell), 8c-cmp (compacted 8-cell), Mo (Morula), Lt-Mo (Late morula), E-Bl (early blastocyst), Ex-Bl (Expanding-Bl), H-Bl (Hatching blastocyst). Antibodies host: Rabbit (Rb), Guinea pig (GP), mouse (m).

Article Snippet: Primary antibodies against RBM47 (HPA006347, Sigma-Aldrich), TJP1 α+ (HP9044; Hycult Biotechnology, Uden, Netherlands) and TJP1 α-(HP9045; Hycult Biotechnology) were diluted and incubated with the embryos overnight at 4°C.

Techniques: Expressing, Proximity Ligation Assay

Journal: Alcoholism, clinical and experimental research

Article Title: Specifically-sized hyaluronan (35 kDa) prevents ethanol-induced disruption of epithelial tight junctions through a Layilin-dependent mechanism in Caco-2 cells

doi: 10.1111/acer.14140

Figure Lengend Snippet: (A/B/C) Caco-2 monolayers were differentiated for 21 days and then treated or not with 100 μg/ml HA35 for 24 hours, followed by challenge with or without 40 mM ethanol for 3 hours. (A) Cells were then fixed in methanol and immunoreactive ZO-1 (green) and occludin (red) visualized by confocal microscopy. Images acquired at 63X with a 3X digital magnification and are representative of at least two images per slide from three independent experiments. (B) Occludin and ZO-1 expression was assessed by immunoblot using ß-actin as a loading control. Semi-quantification of protein expression was performed using Carestream Imaging Software. (C) FITC-dextran (4Kd) was added to the upper chamber of the transwells and leakage into the lower chamber assessed at 1 and 2h. (D) Differentiated Caco-2 monolayers were treated with increasing concentrations of HA35 for 24 h and then challenged with 500 mM ethanol. TEER was measured immediately before the addition of ethanol and 3 h later. Values are expressed as means ±SEM, (A) n=3, (B) n=4–6 and (C) n=12 (values with different superscripts are significantly different within a time point, p<0.05) and (D) n=4 (*p < 0.05 compared to cells not treated with HA35)

Article Snippet: Primary antibodies were purchased from the following sources: rabbit anti-ZO-1 (Life Technologies, 61–7300), guinea pig anti-occludin (Hycult HP9047), rabbit anti-ß-actin (Cell Signaling, 4967), rabbit anti-Layilin (Bioss, 1842R).

Techniques: Confocal Microscopy, Expressing, Western Blot, Imaging, Software

Journal: Alcoholism, clinical and experimental research

Article Title: Specifically-sized hyaluronan (35 kDa) prevents ethanol-induced disruption of epithelial tight junctions through a Layilin-dependent mechanism in Caco-2 cells

doi: 10.1111/acer.14140

Figure Lengend Snippet: Caco-2 monolayers were differentiated on collagen-coated transwell filters and then transfected with scrambled siRNA or siRNA targeting Layilin. (A) Immunofluorescent staining for Layilin 24 h following transfection with scrambled siRNA or siRNA targeting Layilin. Images are representative of at least two images per slide from three independent transfections. (B) 24 h after transfection, cells were treated or not with 100μg/ml HA35 for 24 hours and then challenged with or without 40mM ethanol for 3 hours. Immunoreactive ZO-1 (green) and occludin (red) was visualized by confocal microscopy in cells transfected with scrambled siRNA or siRNA targeting Layilin. Images acquired at 63X with a 3X digital magnification and are representative of at least two images per slide from four independent experiments. (C) Cells transfected with scrambled siRNA or siRNA targeting Layilin were treated with 1 mg/ml HA35 for 24 h and then challenged with 500 mM ethanol. TEER was measured immediately after the addition of ethanol and 3 h later. Values are expressed as means ± SEM. Values are expressed as means ±SEM, (A) n=3, (B) n=6 and (C) n=4–6, *p < 0.05 compared to cells not treated with ethanol within a transfection group.

Article Snippet: Primary antibodies were purchased from the following sources: rabbit anti-ZO-1 (Life Technologies, 61–7300), guinea pig anti-occludin (Hycult HP9047), rabbit anti-ß-actin (Cell Signaling, 4967), rabbit anti-Layilin (Bioss, 1842R).

Techniques: Transfection, Staining, Confocal Microscopy

Journal: JPEN. Journal of parenteral and enteral nutrition

Article Title: Faecalibacterium prausnitzii and a Prebiotic Protect Intestinal Health in a Mouse Model of Antibiotic and Clostridium difficile Exposure

doi: 10.1002/jpen.1053

Figure Lengend Snippet: Effects of Clostridium difficile (CD) on tight junction protein and an anion exchanger expression in proximal colon. Mice were treated as described in Figures 1 and ​and2.2. Proximal colon was collected and embedded in optimal cutting temperature medium (OCT) for histology on days 1 and 5 after CD exposure. (A) Occludin (red), zona occludin-1 (ZO-1; green), (B) claudin-3 (green), and (C) Na+/H+ exchanger isoform 3 (NHE3; green) were visualized by immunohistochemistry in sections of proximal colon frozen in OCT. A selected area was cropped and enlarged. All images were acquired using a 40 × objective. Images are representative of at least replicate images captured per mouse in 3–6 mice per treatment group. FP, Faecalibacterium prausnitzii; PS, potato starch.

Article Snippet: Antibodies were purchased from the following sources: anti–Na + /H + exchanger isoform 3 (NHE 3 ), anti–zona occludin-1 (ZO-1), anti–sodium-coupled monocarboxylate transporter 1 (SLC5A8), and anti–frizzled class receptor 7 (FZD7) from Abcam (Cambridge, MA); anti-occludin from Hycult Biotechnologies (Plymouth Meeting, PA); anti-claudin-3, Alexa Fluor 488, and 568 IgG from Invitrogen (Camarillo, CA).

Techniques: Expressing, Immunohistochemistry