Additional file 1 of Human umbilical cord-derived mesenchymal stem cell transplantation supplemented with curcumin improves the outcomes of ischemic stroke via AKT/GSK-3β/β-TrCP/Nrf2 axis
Creators: name: Li, Yuan
name: Huang, Jialu
name: Wang, Jie
name: Xia, Simin
name: Ran, Hong
name: Gao, Lenyu
name: Feng, Chengjian
name: Gui, Li
name: Zhou, Zhenhua
name: Yuan, Jichao
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Marine Biology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1: Cytotoxicity of curcumin on microglia and hUC-MSC. To determine the optimal concentration of curcumin, we assessed the cytotoxicity of curcumin on microglia and hUC-MSCs after treatmentwith different concentrations of curcumin for 24 h by the CCK-8. For the microglia,there were significant differences in viability when the concentration ofcurcumin was ≥ 16 μmol/L compared with that of the control group, indicating nocytotoxicity when the concentration of curcumin was ≤ 8 μmol/L. For the hUC-MSC, there was no cytotoxicity when the curcumin concentration was ≤ 4μmol/L. Therefore, we selected 4 μmol/L as the optimum concentration ofcurcumin for subsequent experiments. N = 5; N.S., no significant difference, *P &lt; 0.05. Figure S2: Characterization ofhUC-MSCs. The flow cytometry revealed that the phenotypes of mesenchymal stemcells were positive for CD73, CD90 and CD105, while the phenotypes ofhematopoietic stem cells were negative for CD34, CD45 and HLA-DR. Figure S3: Tracing the migration of the hUC-MSC in the CNS. (A) Representative images of immunofluorescence analysis for hUC-MSC identification, hUC-MSC labeled with MAB1281, STRO-1 and CD44, respectively. (B-D) Quantitative analysisof MAB1281+ cells (B), STRO-1+ cells (C) and CD44+cells (D) in the ipsilateral peri-infarct. Cur: curcumin, scale bars = 50 μm,N=3, ***P &lt; 0.001. Figure S4: Combined curcumin-hUC-MSC treatment promoted anti-inflammatory phenotype microglia in MCAO mice. (A-B) Representative images of immunofluorescence analysis formicroglia polarization, (A) The microglial cell-specific marker Iba1 (green), pro-inflammatorymicroglia marker CD86 (red), nuclei (blue); (B) the microglial cell-specificmarker Iba1 (green), anti-inflammatory microglia marker Arg1 (red), nuclei(blue); (C-D) Quantitative analysis of pro-inflammatory (C) and anti-inflammatory(D) microglia in the ipsilateral peri-infarct. Cur: curcumin, scale bars = 20μm; N = 5; N.S., no significant difference, *P &lt; 0.05, **P &lt; 0.01, ***P&lt; 0.001. Figure S5: Nrf2 involvement in the anti-inflammatoryphenotype microglia polarization mediated by combined curcumin-hUC-MSC treatmentin MCAO mice. (A-C) Representative flow cytometric (A) and quantitative datafor the percentage of CD86+ cells (B) and CD206+ (C) microglia in the ipsilateral peri-infarct isolated from MCAO mice subjected todifferent treatments. (D-G) qRT-PCR analysis of pro-inflammatory microglia gene iNOS (D) and CD86 (E) and anti-inflammatory microglia gene Arg1 (F) and CD206 (G). Combined curcumin-hUC-MSC treatment decreased the pro-inflammatory marker expressionand increased anti-inflammatory marker expression. Inhibition of AKT with MK2206 or activation of GSK3β with SNP increased pro-inflammatory subtype marker expression and decreased the anti-inflammatory marker expression, whereas further suppression of GSK3β (MK2206+TDZD-8) enhanced anti-inflammatory marker expression at both protein and mRNA levels. Furthermore, Nrf2 knockout abolished the anti-inflammatorymicroglia phenotypic polarization mediated by the combined treatment. Cur,curcumin, N = 5; N.S., no significant difference, **P &lt; 0.01, ***P &lt;0.001.