Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: 9-cis-retinoic acid signaling in Sertoli cells regulates their immunomodulatory function to control lymphocyte physiology and Treg differentiation

doi: 10.1186/s12958-024-01246-2

Figure Lengend Snippet: Effect of 9cRA on the expression of pro-inflammatory ( A - A ”) and anti-inflammatory ( B - B ”) genes and proteins in murine Sertoli cells. Cells were treated with a vehicle (control, C), or 10 − 7 M 9cRA for 24 h. ( A , B ) Relative expression of Ifng, Tnfr1, Il1a, Il6, Tgfb1, Il10, Gal1 , and Ido mRNAs was determined using quantitative real-time RT–PCR analysis. The histograms are the quantitative representation of data of three independent experiments, each in triplicate. The expression of the individual genes was normalized to the mean expression of the reference genes ( Rn18s, B2m, Rpl13a , and Hprt1 ) as an internal control (relative quantification, RQ). ( A’ , B’ ) Western blot detection of TGFβ, IL-10, GAL-1, IDO, IFN-γ, TNFR1, IL-1α, and IL-6 proteins. The histograms are the quantitative representation after densitometry of data (mean ± SD) of three independent experiments, each in triplicate. The relative level of studied protein was normalized to β-actin. The protein levels within the control group were arbitrarily set as 1. Significant differences from control values are denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001 ( A” , B” ) Subcellular localization of TGFβ, IL-10, GAL-1, IDO, IFN-γ, TNFR1, IL-1α, and IL-6 proteins in murine Sertoli cells was visualized by immunofluorescence. Scale bar = 100 μm

Article Snippet: Anti-GAL-1 , Rabbit , ABclonal , A4732 , 1:500 (WB); 1:100 (IF).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Gut pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis.

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Fig. 1 In silico predicted conserved murine and human targets of miR-125a-5p and miR-615-3p. A The Venn diagram presents interfaces of overlapping targets of miR-125a-5p and miR-615-3p within the mucin-type O-glycosylation pathway of which the glycosyltransferases St3gal1 and B4galt1 are conserved in human and mouse, respectively. B Excerpt of the KEGG signalling pathway enrichment analysis of mucin-type O-glycosylation in mouse and human [20]. Marked by the red asterisks are the specific steps in the glycosylation that might be modified by miR-125a-5p and miR-615-3p interaction. C Two binding sites of miR-125a-5p were identified in the 3’UTR of the murine St3gal1 using RNAhybrid [21]

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: In Silico, Glycoproteomics, Modification, Binding Assay

Journal: Gut pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis.

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Fig. 2 Specific interaction between miR-125a-5p and St3gal1 was verified by RNAi and dual luciferase reporter assay. A Transfection efficiency of the murine intestinal cell line CMT 93 with miR-125a-5p mimics as well as expression of potential targets was evaluated by means of RT-qPCR. Most pronounced and significant decrease of the target St3gal1 was detected after miR-125a-5p transfection. B4galt1 showed significantly reduced levels. St3gal2 showed no significant difference. miR-615-3p showed upregulated but not significant expression. Non-target miRNA was used as a control, fold changes were calculated relatively to the non-target control and normalised with HPRT and SDHA or SNORD44 and SNORD 47, respectively. Charts indicate means ± standard deviations (SD) of three biological samples with triple measurements. B Relative luciferase activity was determined in comparison to control non-target miRNA mimic. miR-125a-5p caused clear and significant activity of the reporter gene fused to the target sites compared with non-target transfected controls. Charts indicate means ± SD of three biological replicates with technical triplicates. Statistical significance is presented by asterisks compared to negative controls at each time point. *P ≤ 0.05, **P ≤ 0.01, unpaired t-test

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR, Control, Activity Assay, Comparison

Journal: Gut pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis.

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Fig. 3 Relative gene expression of relevant miRNAs and their target mRNAs in secondary abiotic IL10 −/− mice colonic tissue sections six days post C. jejuni infection. 19 C. jejuni infected and 20 naïve control mice were included in the study. Each data point in the diagram represents an animal that showed confirmed expression in the respective analysis. A Upon C. jejuni infection, expression of miR-125a-5p was significantly decreased, whereas transcriptional levels of St3gal1 and B4galt1 were significantly enhanced. B miR-615-3p levels were significantly increased, while St3gal2 was significantly downregulated. Expressions were relatively calculated to naïve controls and normalised with SNORD44 and SNORD47 or with HPRT and SDHA, respectively. For each individual, the mean was calculated based on triplicate measurements. Charts show mean ± SD of all individuals. Statistical significance is presented by asterisks compared to naïve controls. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired t-test

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: Gene Expression, Infection, Control, Expressing

Journal: Gut pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis.

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Fig. 4 Increased colonic ST3GAL1 protein levels in secondary abiotic IL10 −/− mice six days post C. jejuni infection compared to naïve controls. A Increased ST3GAL1 protein levels were detected by western blots in pooled protein samples of infected mice. GAPDH is presented as the respective loading reference. B ST3GAL1 detection of eight C. jejuni infected and eight naïve mice colon samples were quantified by means of western blotting followed by densitometric analysis relative to the respective GAPDH signals as controls. Protein levels of ST3GAL1 in the infected group were significantly and 1.7-fold increased, compared to naïve controls. Charts show normalised mean ± SD in each group. Statistical significance is presented by asterisks. **P ≤ 0.01, unpaired t-test. C Localisation and enhanced ST3GAL1 expression in the colon of C. jejuni infected mice determined in representative immunofluorescent staining compared to naïve controls. ST3GAL1 was detected by immunofluorescent staining and shown in red using DyLight 594 whereas the nuclei were stained blue using DAPI. Enhanced red signal intensity and pronounced cytosolic distribution was detected in infected controls. The top row shows the overview, with the area outlined in green in the bottom row shown enlarged. Scale bars indicate 100 µm and 50 µm. Exposure time was identical for all colon sections and presented images are representative for three biological replicates tested

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: Infection, Western Blot, Expressing, Staining