Journal: Microbiology spectrum

Article Title: Phillyrin ameliorates DSS-induced colitis in mice via modulating the gut microbiota and inhibiting the NF-κB/MLCK pathway.

doi: 10.1128/spectrum.02006-24

Figure Lengend Snippet: Figure 5A illustrates that p-NF-κB p65 and p-IκB expression in the DSS group was markedly enhanced in comparison to the control group. However, the PHY group notably reversed these effects in comparison to the DSS group (Fig. 6A through C). Research has demonstrated that NF-κB can enhance MLCK transcription, increasing MLC phosphory lation and decreasing TJ protein expression (23). Furthermore, the MLCK and p-MLC protein expression in DSS was enhanced in comparison to the control group. Neverthe less, in the PHY pre-protected group, the expression of MLCK (Fig. 6A and D) and p-MLC (Fig. 6A and E) proteins was lower in comparison to the DSS group. These outcomes revealed that PHY could restore the intestinal barrier by regulating the NF-κB /MLCK/MLC signaling pathway.

Article Snippet: The protein was transferred from gel to polyvinylidene difluoride membrane (PVDF) (Millipore, Darmstadt, Germany) by wet transfer method at constant flow for 220 mA and 75 min. After the transfer, 5% skim milk powder was added, and the table was closed at room temperature for 4 h. After closure, tris-buffered saline with Tween 20 (TBST) was washed three times for 10 min each time, and then the primary antibody was incubated in primary antibodies against occludin (1:1,000; Proteintech), myosin light-chain kinase (MLCK) (1:1,000; Abcam, Cambridge, UK), phosphorylated myosin light chain (p-MLC) (1:1,000; Abcam), myosin light-chain (MLC) (1:1,000; CST, Boston, USA), p-NF-κB (1:1,000; Abcam), NF-κB (1:1,000; Abcam), p-IκB (1:1,000; CST), IκB (1:1,000; CST), claudin-3 (1:1,000; Abcam), inducible nitric oxide synthase (iNOS) (1:1,000; Proteintech), cyclooxygenase-2 (COX-2) (1:1,000; Proteintech), and β-actin (1:1,000; Proteintech) at 4°C overnight.

Techniques: Expressing, Comparison, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Exosomes derived from fibroblasts in DFUs delay wound healing by delivering miR-93-5p to target macrophage ATG16L1.

doi: 10.1016/j.bbadis.2024.167640

Figure Lengend Snippet: Fig. 3. miR-93-5p affects macrophage autophagy function by affecting Atg16L1. (A) Nine mRNAs were identifed based based on TargetScan, miRDB, miRTarBase and Human Autophage Database. (B) Schematic representation of the wild-type and mutant-type binding site between the 3′UTR of ATG16L1 and miR-93-5p. (C) Relative luciferase activity of 3′UTR-ATG16L1-luc constructs in HEK293T cells after transfection of miR-93-5p mimics/NC; n = 3 per group. (D) The protein levels of ATG16L1 in bone marrow-derived macrophages transfected with miR-93-5p NC/mimics or PBS (Control) by Western blot. (E) The protein levels of ATG16L1 in skin tissues from normal or diabetic wound (DFUs) by Western blot. (F) Immunofluorescence staining for ATG16L1 (red) and F4/80 (green) in skin tissues from normal or diabetic wound (DFUs); scale bar, 100 μm. (G) The protein levels of LC3b and P62 in BMDMs transfected with miR-93-5p NC/mimics or PBS by Western blot, immunofluorescence staining for LC3b (green) in bone marrow-derived macrophages transfected with miR-93-5p NC/mimics or PBS; scale bar, 50 μm. (H) The protein levels of ATG16L1, LC3 and P62 in fibroblast-derived exosomes treated bone marrow-derived macrophages transfected with miR-93-5p inhibitor NC/in hibitor or PBS (Control) by Western blot. Data are expressed as mean ± SD, ns P > 0.05, **P < 0.01 (unpaired t-test or ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Next, sections were incubated overnight at 4 ◦C with primary antibodies against F4/80 (Servicebio, GB11027), Atg16L1 (CST, #8089), LC3B (Proteintech, 81004-1-RR), α-SMA (Proteintech,14395-1AP) and Collagen Type I (Proteintech, 14695-1-AP).

Techniques: Mutagenesis, Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Derivative Assay, Control, Western Blot, Immunofluorescence, Staining

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Berberine alleviates AGEs-induced ferroptosis by activating NRF2 in the skin of diabetic mice.

doi: 10.3389/ebm.2024.10280

Figure Lengend Snippet: FIGURE 4 BBR protected HaCaT cells against AGEs-induced ferroptosis partially through NRF2 activity. (A) Representative Western blot experiments illustrating NRF2 protein expression in HaCaT cells after transfection with NRF2 siRNA. (B) The mock-, NRF2 siRNA- or Con siRNA-transfected HaCaT cells were cultured with 150 μg/mL AGEs and 5 μM BBR for 48 h, and cell viability was examined using CCK-8 assays. (C) The relative levels of MDA were measured in HaCaT cells after treatment. (D) Representative images and quantitative analysis of the protein levels of GPX4 and FTL in HaCaT cells. The results are expressed as the mean ± SD. Data are representative of three independent experiments. *P ≤0.05, **P ≤0.01. AGEs, advanced glycation end products; BBR, berberine; Con siRNA, control siRNA; FTL, ferritin light chain; GPX4, glutathione peroxidase 4; MDA, malondialdehyde; NRF2, nuclear factor E2-related factor 2; ns, no significance.

Article Snippet: After blocking for 1 h at room temperature, the membranes underwent an overnight incubation with primary antibodies against the following proteins: glutathione peroxidase 4 (GPX4; 1:1000; 67763-1-Ig; Proteintech, Wuhan, China), ferritin light chain (FTL; 1:1000; 10727-1-AP; Proteintech), NRF2 (1:800; 12721; Cell Signaling Technology, Danvers, United States), α-tubulin (1: 20,000; HRP-66031; Proteintech), or lamin B1 (1:10,000; 12987- 1-AP; Proteintech).

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Cell Culture, CCK-8 Assay, Control