Journal: Journal of Pain Research

Article Title: Metabolomics Analysis Reveals Gut Microbiota-Associated Sakuranin Modulates Endometrial Stem Cell Differentiation and Inflammation to Alleviate Pain in Endometriosis

doi: 10.2147/JPR.S557430

Figure Lengend Snippet: Sakuranin inhibits the differentiation of EnSCs into mesenchymal cells and reduces the levels of inflammatory factors. ( A ) The expression level of the Sushi Domain-containing 2 (SUSD2) protein in EnSCs. The expression of the SUSD2 protein is presented in red fluorescence, and the nuclear DAPI staining is shown in blue; ( B ) The flow cytometry scatter plot of EnSCs without treatment with the SUSD2 antibody (left) and of EnSCs incubated with the SUSD2 antibody (right); ( C ) Screening for the optimal concentration of sakuranin; ( D – F ) Detection of the contents of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in EnSCs by enzyme-linked immunosorbent assay (ELISA); ( G – I ) Detection of the expression levels of the activated leukocyte cell adhesion molecule (ALCAM) and the vascular endothelial growth factor (VEGF) in EnSCs by Western blot and the quantitative analysis of protein expression; ( J – M ) Detection of the mRNA expressions of epithelial cadherin (E-cadherin), cytokeratin 10 (Cytokeratin 10), vimentin, and neural cadherin (N-cadherin) in human primary EnSCs by real-time quantitative polymerase chain reaction (RT-qPCR); ( N ) Phalloidin staining of the cytoskeleton of EnSCs. The green fluorescence represents phalloidin staining, and the blue fluorescence represents the counterstaining of the nucleus by DAPI; ( O ) Detection of the migratory ability by scratch assay and the quantitative analysis of the scratch healing of EnSCs. The cells were treated with 1 μg/mL lipopolysaccharide (LPS) for 48 hours. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After the transfer, the membranes were blocked at room temperature for 2 hours before overnight incubation with primary antibodies, including VEGF (1:5000, 19003-1-AP, Proteintech); ALCAM (1:5000, 21972-1-AP, Proteintech); GAPDH (1:15000, 60004-1-lg, Proteintech) at 4 °C.

Techniques: Expressing, Fluorescence, Staining, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Wound Healing Assay

Journal: Journal of Pain Research

Article Title: Metabolomics Analysis Reveals Gut Microbiota-Associated Sakuranin Modulates Endometrial Stem Cell Differentiation and Inflammation to Alleviate Pain in Endometriosis

doi: 10.2147/JPR.S557430

Figure Lengend Snippet: Immunofluorescence detection of protein expression in the endometrium of normal mice and the ectopic endometrial tissue of EMS mice. ( A – D ) Tissue immunofluorescence detection of the expressions of Cluster of differentiation 146 (CD146), ALCAM, VEGF, and Ki67 proteins. The expressions of the detected proteins are presented in red fluorescence, and the nuclear DAPI staining is shown in blue; ( E ) Tissue immunofluorescence detection of the expressions of tyrosine hydroxylase (TH) and the marker substance P (SP). Red represents the expression of SP protein, green represents the expression of TH protein, and blue represents the nuclear staining by DAPI.

Article Snippet: After the transfer, the membranes were blocked at room temperature for 2 hours before overnight incubation with primary antibodies, including VEGF (1:5000, 19003-1-AP, Proteintech); ALCAM (1:5000, 21972-1-AP, Proteintech); GAPDH (1:15000, 60004-1-lg, Proteintech) at 4 °C.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Marker