Journal: Advanced Science

Article Title: Hippo Pathway Activation in Aged Mesenchymal Stem Cells Contributes to the Dysregulation of Hepatic Inflammation in Aged Mice

doi: 10.1002/advs.202300424

Figure Lengend Snippet: Downregulation of YAP1 in aged MSCs leads to reduced immunosuppressive function. A GSEA analysis of gene expression of young and aged MSCs shows that Hippo signaling is upregulated in aged MSCs. B) mRNA expression levels of Yap1 and YAP1 target genes were tested by RT‐PCR. C,D) Cytosolic and nuclear expression of YAP1 was detected by western blotting and quantified relative to the internal controls β‐ACTIN and Histone H3. E,F) Translocation of YAP1 into the nucleus was detected in young and aged MSCs by immunofluorescence staining and corresponding quantification. G) YAP1 expression was tested in young and aged liver MSCs by western blotting. β‐ACTIN was used as the internal control. H,I) IHC staining and quantification of CD8 + T‐cells in the liver. J,K) H&E staining of the liver after treatment with young and aged MSCs, with or without manipulation of Yap1 expression, and quantification of the necrotic areas. L) Measurement of serum ALT and AST levels in the indicated mice. Y‐MSCs, young MSCs; A‐MSCs, aged MSCs. N.D., not detected. Data (B, D, F) were analyzed using two‐tailed unpaired Student t ‐test. Data (I, K, L) were analyzed using one‐way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Graphs showed mean ± S.D. N = 3, the experiments were repeated for three times.

Article Snippet: A Bestar qPCR RT Kit (DBI Bioscience, Ludwigshafen, Germany) was used for reverse transcription to cDNA, and real‐time PCR was performed using a SYBR PrimeScript RT‐PCR Kit (DBI bioscience, Ludwigshafen, Germany) with an ABI Prism 7300 system.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Translocation Assay, Immunofluorescence, Staining, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: Hippo Pathway Activation in Aged Mesenchymal Stem Cells Contributes to the Dysregulation of Hepatic Inflammation in Aged Mice

doi: 10.1002/advs.202300424

Figure Lengend Snippet: YAP1 maintains the immunosuppressive activity of MSCs by promoting iNOS expression. A) mRNA expression levels of genes encoding anti‐inflammation factors were detected in young and aged MSCs by RT‐PCR after treatment with I+T for 0 h, 4 h, and 8 h. B) Expression of iNOS protein in young and aged MSCs was detected by western blotting assay at 0, 24, and 48 h after I+T treatment and corresponding quantification. β‐ACTIN was used as the internal control. C, Nitrate concentrations were measured by a Griess reagent kit in conditioned medium of young and aged MSCs after I+T treatment. D,E) IHC staining of CD8 positive cells in the liver after treatment with young and aged MSCs (with or without manipulation of Nos2 expression) and corresponding quantification. F,G) H&E staining of the liver after treatment with young and aged MSCs (with or without manipulation of No` expression) and quantification of the necrotic areas. H,I) Young MSCs were transfected by shYap1 adenovirus, and aged MSCs were transfected by Yap1 overexpression adenovirus. iNOS expression was detected by western blotting assay in young and aged MSCs treated with I+T for the indicated time. Y‐MSCs, young MSCs; A‐MSCs, aged MSCs. N.D., not detected. Data (A, B, C) were analyzed using two‐tailed unpaired Student t ‐test. Data (E, G) were analyzed using one‐way ANOVA test. *** p < 0.001, **** p < 0.0001. Graphs showed mean ± S.D. N = 3, the experiments were repeated for three times.

Article Snippet: A Bestar qPCR RT Kit (DBI Bioscience, Ludwigshafen, Germany) was used for reverse transcription to cDNA, and real‐time PCR was performed using a SYBR PrimeScript RT‐PCR Kit (DBI bioscience, Ludwigshafen, Germany) with an ABI Prism 7300 system.

Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Transfection, Over Expression, Two Tailed Test

Journal: Advanced Science

Article Title: Hippo Pathway Activation in Aged Mesenchymal Stem Cells Contributes to the Dysregulation of Hepatic Inflammation in Aged Mice

doi: 10.1002/advs.202300424

Figure Lengend Snippet: YAP1 promotes iNOS expression by binding the Stat1 promoter and enhancing Stat1 transcription. A, B, Young MSCs were transfected by shYap1 adenovirus and then treated with I+T for 0, 30, and 60 min. Aged MSCs were transfected by Yap1 overexpression adenovirus and then treated with I+T for 0, 30, and 60 min. Expression of YAP1, STAT1, and p‐STAT1 (Ser727) was detected by western blotting. β‐ACTIN was used as the internal control. C,D) Detection of iNOS expression by western blotting assay in young and aged MSCs in the rescue experiment. β‐ACTIN was used as the internal control. E) The mRNA expression levels of Stat1 in young and aged MSCs were detected by RT‐PCR after adenovirus‐mediated manipulation of Yap1 expression. F) Young MSCs were treated with and without 10 µM VP for 24 h. The levels of STAT1, CTGF, and CYR61 were detected by western blotting assay. β‐ACTIN was used as the internal control. G) CUT&Tag assay was performed to verify the target motif bound by YAP1/TEADs in the Stat1 promoter. Anti‐IgG was used as the negative control. Y‐MSCs, young MSCs; A‐MSCs, aged MSCs. Data were analyzed using two‐tailed unpaired Student t ‐test. ** p < 0.01, *** p < 0.001. The graph showed mean ± S.D. N = 3, the experiments were repeated for three times.

Article Snippet: A Bestar qPCR RT Kit (DBI Bioscience, Ludwigshafen, Germany) was used for reverse transcription to cDNA, and real‐time PCR was performed using a SYBR PrimeScript RT‐PCR Kit (DBI bioscience, Ludwigshafen, Germany) with an ABI Prism 7300 system.

Techniques: Expressing, Binding Assay, Transfection, Over Expression, Western Blot, Reverse Transcription Polymerase Chain Reaction, Negative Control, Two Tailed Test