4322t  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Sirt6 attenuates chondrocyte senescence and osteoarthritis progression

doi: 10.1038/s41467-022-35424-w

Figure Lengend Snippet: a , b Heat map ( a ) and Volcano plot ( b ) demonstrating differentially expressed genes (fold change >2 or <0.5, Benjamini–Hochberg-corrected p ) in Sirt6 KO chondrocytes vs controls. Notably, STAT5, JAK3, and IL-15 were observed to be significantly upregulated. c GO analysis of upregulated genes from Sirt6 KO chondrocytes for biological processes (BP), cellular component (CC), and molecular function (MF). d KEGG and GSEA analysis demonstrating JAK/STAT signaling pathway enriched in OA. e The rescue experiments were performed in human OA chondrocytes to validate the relationship between Sirt6 and IL-15/JAK3/STAT5 signaling pathway. pcDNA3.1-Sirt6, Sirt6 siRNA, or pcDNA3.1-Sirt6 + pcDNA3.1-STAT5 was transfected into human OA chondrocytes. After 48 h, the related genes were analyzed. n = 3 independent biological replicates per group. f – h Human chondrocytes were infected with Ad-control or Ad-Sirt6, then treated with or without IL-15 (2 ng/mL) for the indicated time. n = 3 independent biological replicates per group ( f ). Immunofluorescent staining of cellular Stat5 and nuclear and cytoplasm fraction of Stat5 ( g ). n = 3 independent biological replicates per group. Western blot analysis of p-Stat5 and Stat5 expression level. n = 3 independent biological replicates per group ( h ). i Human chondrocytes were transfected with Stat5, then treated with or without IL-15 (2 ng/mL). Whole-cell lysates were immunoprecipitated with anti-Sirt6 or anti-Stat5 antibodies, and precipitated proteins were detected by anti-Stat5 or anti-Sirt6 antibodies, respectively. Western blot detection of nuclear acetylation level of Stat5 in human chondrocytes with or without IL-15 treatment. n = 3 independent biological replicates per group. j Human chondrocytes were transfected with the Stat5 reporter and Stat5 with or without Sirt6, then treated with or without IL-15 for 12 h. Luciferase activity was normalized to β-gal. n = 6 independent biological replicates per group. k ChIP assay analysis of human chondrocytes infected with Ad-control or Ad-Sirt6, then treated with or without IL-15. n = 6 independent biological replicates per group. Scar bar: g 20 μm. Data were presented as the mean ± s.e.m. P values are from two-tailed Mann–Whitney U -test ( a – d ), one-way ANOVA test followed by Tukey’s post hoc test ( e , g ) or Brown–Forsythe and Welch ANOVA test followed by Tamhane’s T2 post hoc analysis ( j , k ). Source data are provided as a Source Data file.

Article Snippet: After washing three times with PBS (5 min each time), cells were blocked by blocking solution (5% BSA in TBS with 0.5% Triton X-100) for 2 h for the following incubation with primary antibodies, p16 INK4a (1:1000, Cell Signaling Technology, #18769), TNF-α (1:500; Abcam, ab1793), IL-6 (1:1000; Abcam, ab246703), IL-1β (1:100, Abcam, ab156791), P21 (1:800, Cell Signaling Technology, #2947), p53 (1:1000, Cell Signaling Technology, #2527), HMBG1 (1:2000; Abcam, ab18256), p-JAK3 (1:1000; Abcam, ab45141), STAT5 (1:200, Cell Signaling Technology, #25656), p-STAT5 (1:100, Cell Signaling Technology, #4322) and Alexa Fluor 555 (1:100, Abcam, ab150078)- or Alexa Fluor 488 (1:1000, Abcam, ab150077)-conjugated secondary antibodies and DAPI (Invitrogen).

Techniques: Transfection, Infection, Control, Staining, Western Blot, Expressing, Immunoprecipitation, Luciferase, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Sirt6 attenuates chondrocyte senescence and osteoarthritis progression

doi: 10.1038/s41467-022-35424-w

Figure Lengend Snippet: a LC-MS/MS analysis identified Sirt6 deacetylated K163 on Stat5. Lysates from human chondrocytes with or without Sirt6 treatment. b Interaction model between Stat5 and Sirt6. c Conservation of Stat5 K163 in different species. d K163 is located in exon 5 of STAT5, which was mutated to arginine through “A” replaced by “G”. The surface from molecular simulation showed the mutant site (K163R). e Sirt6 deacetylated Stat5 via K163. The acetylation level of WT Stat5 with Sirt6 treatment was analyzed by western blot. n = 3 independent biological replicates per group. f , g Human chondrocytes were transfected with WT Stat5 or mutant Stat5, followed by IL-15 treatment. The phosphorylation of Stat5 and the cellular localization of Stat5 was determined by western blot. n = 3 independent biological replicates per group ( f ). Immunofluorescent staining of Stat5. n = 3 independent biological replicates per group ( g ). h Transcriptional activity of WT and mutant Stat5. Human chondrocytes were co-transfected with WT Stat5 or mutant Stat5, followed by IL-15 treatment. n = 6 independent biological replicates per group. i , j K163 of Stat5 is required for Sirt6-regulated IL-15/JAK3/STAT5 signaling pathway. After IL-15 treatment, the phosphorylation of Stat5 and the cellular localization of Stat5 were analyzed. n = 3 independent biological replicates per group ( i ). Immunofluorescent staining of Stat5. n = 3 independent biological replicates per group ( j ). k The transcriptional activity of WT and mutant Stat5 in human chondrocytes transfected by pcDNA3.1 or pcDNA3.1-Sirt6, followed by IL-15 treatment. n = 6 independent biological replicates per group. l Molecular model for dysregulated Sirt6 in chondrocyte senescence and OA pathogenesis. Scar bar: g , j 20 μm. Data were presented as the mean ± s.e.m. P values are from two-tailed unpaired Student’s t -test ( h , k ). Source data are provided as a Source Data file.

Article Snippet: After washing three times with PBS (5 min each time), cells were blocked by blocking solution (5% BSA in TBS with 0.5% Triton X-100) for 2 h for the following incubation with primary antibodies, p16 INK4a (1:1000, Cell Signaling Technology, #18769), TNF-α (1:500; Abcam, ab1793), IL-6 (1:1000; Abcam, ab246703), IL-1β (1:100, Abcam, ab156791), P21 (1:800, Cell Signaling Technology, #2947), p53 (1:1000, Cell Signaling Technology, #2527), HMBG1 (1:2000; Abcam, ab18256), p-JAK3 (1:1000; Abcam, ab45141), STAT5 (1:200, Cell Signaling Technology, #25656), p-STAT5 (1:100, Cell Signaling Technology, #4322) and Alexa Fluor 555 (1:100, Abcam, ab150078)- or Alexa Fluor 488 (1:1000, Abcam, ab150077)-conjugated secondary antibodies and DAPI (Invitrogen).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Western Blot, Transfection, Phospho-proteomics, Staining, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Sirt6 attenuates chondrocyte senescence and osteoarthritis progression

doi: 10.1038/s41467-022-35424-w

Figure Lengend Snippet: a SA-β-Gal positivity was analyzed in human OA chondrocytes after 3 and 4 weeks of culture with tgg2-PP-MDL-800 NP or control (5, 10, or 20 µM). n = 6 independent biological replicates per group. b Representative immunofluorescent images of indicated markers in human OA chondrocytes after 4 weeks of culture with tgg2-PP-MDL-800 NP or control (5, 10, or 20 µM). n = 6 independent biological replicates per group. c Proteoglycan content was measured by safranin O-fast green staining in human OA cartilage explants after 3 and 4 weeks of incubation with tgg2-PP-MDL-800 NP. n = 6 independent biological replicates per group. d Western blot were performed to determine the Col II expression level at 3 and 4 weeks. n = 3 independent biological replicates per group. e Overview of the experimental set-up with injections of PBS, tgg2-PP NP or tgg2-PP-MDL-800 NP. f Representative immunofluorescent images of IL-15, p-JAK3, and p-STAT5 levels in these chondrocytes from different treatment groups. n = 6 independent biological replicates per group. g Western blot analysis of protein levels of Acetyl, p-Stat5 (Y694), and Stat5. n = 3 independent biological replicates per group. h , i At 4 and 8 weeks, Safranin O-fast green stained sections and H&E of cartilage (medial femoral condyle) showed that OA phenotype was significantly alleviated in OA mice model treated by tgg2-PP-MDL-800 NP, as evaluated by OARSI. n = 6 mice per group ( h ). A high level of Col II was observed in the tgg2-PP-MDL-800 NP treatment group. n = 3 independent biological replicates per group ( i ). j At 4 and 8 weeks, synovial inflammation was evaluated. n = 6 mice per group. k Representative micro-CT images of subchondral bone in the medial tibial plateau of PBS, tgg2-PP NP, or tgg2-PP-MDL-800 NP-treated DMM mice at 4w and 8w. n = 6 mice per group. Scar bar: c 200 μm, h , j (upper) 50 μm, a , b , j (lower) 20 μm, f 10 μm. Data were presented as the mean ± s.e.m ( a – d , i ) or median (25–75th percentiles) ( f , h, j). P values are from two-tailed unpaired Student’s t -test ( a , control and 10 μM groups in c ), two-tailed unpaired t -test with Welch’s correction (5 and 20 μM groups in c ), one-way ANOVA test followed by Tukey’s post hoc ( b ), two-way ANOVA test followed by Tukey’s post hoc ( d , i ), Kruskal–Wallis test followed by Dunn’s post hoc ( f ) or Scheirer–Ray–Hare test followed by Dunn’s post hoc ( h , j ). Source data are provided as a Source Data file.

Article Snippet: After washing three times with PBS (5 min each time), cells were blocked by blocking solution (5% BSA in TBS with 0.5% Triton X-100) for 2 h for the following incubation with primary antibodies, p16 INK4a (1:1000, Cell Signaling Technology, #18769), TNF-α (1:500; Abcam, ab1793), IL-6 (1:1000; Abcam, ab246703), IL-1β (1:100, Abcam, ab156791), P21 (1:800, Cell Signaling Technology, #2947), p53 (1:1000, Cell Signaling Technology, #2527), HMBG1 (1:2000; Abcam, ab18256), p-JAK3 (1:1000; Abcam, ab45141), STAT5 (1:200, Cell Signaling Technology, #25656), p-STAT5 (1:100, Cell Signaling Technology, #4322) and Alexa Fluor 555 (1:100, Abcam, ab150078)- or Alexa Fluor 488 (1:1000, Abcam, ab150077)-conjugated secondary antibodies and DAPI (Invitrogen).

Techniques: Control, Staining, Incubation, Western Blot, Expressing, Micro-CT, Two Tailed Test

Journal: Brain Sciences

Article Title: Regulation of Microglia-Activation-Mediated Neuroinflammation to Ameliorate Ischemia-Reperfusion Injury via the STAT5-NF-κB Pathway in Ischemic Stroke

doi: 10.3390/brainsci12091153

Figure Lengend Snippet: Dauricine inhibited the activation of STAT5, NF-κB and docked with STAT5. ( A ) The western blot representative figure of NF-κB, P-NF-κB, STAT5, P-STAT5 and β-tubulin were showed and was counted with ratio of P-STAT5 to STAT5 ( B ) and P-NF-κB to NF-κB ( C ). n = 3, Control group vs. LPS group, * p < 0.05, ** p < 0.01; LPS group vs. (LPS + Dauricine) group, # p < 0.05, unpaired Student’s t -test. Ligand receptor docking was performed with AutoDock and analyzed with PyMOL. The top panel shows the joint diagram of dauricine and STAT5a protein ( D ), and the lower panel shows the joint diagram of dauricine and STAT5b protein ( E ), the energy data under natural conditions and molecular docking is noted at bottom right.

Article Snippet: Then 5% skim milk was applied to block the membranes for 2 h at room temperature; then, we incubated them at 4 °C for at least 12 h with the following antibodies: anti-Stat5 antibody (CST, 9363S, 1:1000), anti-phospho-Stat5 antibody (CST, 4322S, 1:1000) (endogenous levels of Stat5a only when phosphorylated with Tyr694 and endogenous levels of Stat5b when phosphorylated with Tyr699), anti-P-NF-kB antibody (CST, 3033S, 1:1000), anti-NF-κB antibody (CST, 8242S, 1:1000), anti-β-tubulin antibody (Bioworld Technology, AP0064, 1:2000), and anti-β-actin antibody (Bioworld Technology, AP0060, 1:2000).

Techniques: Activation Assay, Western Blot, Control