Journal: Aging Cell

Article Title: NF‐κB/IKK activation by small extracellular vesicles within the SASP

doi: 10.1111/acel.13426

Figure Lengend Snippet: Small molecule inhibitor screen identifies different signalling pathways mediated by the evSASP (a) Details of the experimental settings to unveil novel signalling pathways mediated by the evSASP. Senescence was induced in iRAS donor cells by addition of 200 nM 4‐hydroxytamoxifen (+4OHT). HFFF2 recipient cells were treated with a panel of small molecule inhibitors in addition to sEV isolated from iC and iRAS cells. Drugs were maintained for 6 days and washed out. (b) Box and Whiskers plot representing the data obtained from the screen measuring proliferation by quantifying the percentage of HFFF2 that stain positive for BrdU. The graph indicates the targets of the small molecule inhibitors used: 40 µM PD98059 (targeting MEK1/2), 20 µM SB202190 (p38MAPK), 4 µM TGFB‐R1 (TGFBR1 kinase), 8 µM VEGFR2 (VEGFR2), 150 nM GSK429286A (ROCK1/2, Rho‐associated kinase), 50 nM CPD22 (ILK, integrin‐linked kinase), 1 µM CPG (MNK1/2), 100 nM TORIN2 (mTOR, mammalian target of rapamycin), 1µM RUXOLITINIB (JAK1/2 inhibitor), 40µM AG‐490 (JAK2/3 kinase), 45 µM JANEX1 (JAK3 kinase), 1 µM AG‐879 (protein Tyrosine Kinase), 2 µM IMATINIB (tyrosine kinase; TK1), 20 µM CAY10576 (IKKε, IκB kinase epsilon), 1.5 µM SUNITINIB (multi tyrosine kinase, multi‐TK). Data show the min to max Box and Whiskers plot of 4 independent experiments. Dunnett's multiple comparison test analysis was performed to determine statistical significance. p‐values are shown. (c) Representative IF pictures for BrdU staining in HFFF2 cells treated with or without 20 µM CAY10576 (IKKε inhibitor) and sEV from iC or iRAS. Scale bar: 50 µm. (d) Representative IF images (left panels) and quantification (right panels) of cells staining positive for senescence‐associated beta galactosidase activity (SA‐β‐Gal) in HFFF2 fibroblasts treated with 20 µM CAY10576 and sEV derived from iC or iRAS cells. Scale bar of IF pictures: 50 µm. All data represent the mean ± SEM of 4–5 independent experiments. Data were normalized to sEV from iRAS sample. One‐way ANOVA analysis was performed with multiple comparisons to the sEV iRAS control sample

Article Snippet: HFFF2 recipient cells were treated with several inhibitors at the below concentrations simultaneously with sEV in medium supplemented with 10% (v/v) EV‐depleted FBS and 1% (v/v) A/A for 72 h. Treatment with inhibitors and sEV was repeated 72 h later and readout was determined after 72 h. Small molecule inhibitor concentrations used for the screen: 40 µM PD98059 (targeting MEK1/2; ThermoFisher), 20 µM SB202190 (p38MAPK; Santa Cruz Biotech), 4µ M TGFB‐R1 (TGFBR1 kinase; Calbiochem), 8 µM VEGFR2 (VEGFR2; Calbiochem), 150 nM GSK429286A (ROCK1/2, Rho‐associated kinase; Abcam), 50 nM CPD22 (ILK, integrin‐linked kinase; Calbiochem), 1 µM CPG (MNK1/2; Calbiochem), 100 nM TORIN2 (mTOR, mammalian target of rapamycin; CAYMAN Chemical), 1µM RUXOLITINIB (JAK1/2 inhibitor; CAYMAN Chemical), 40 µM AG‐490 (JAK2/3 kinase; CAYMAN Chemical), 45 µM JANEX1 (JAK3 kinase; CAYMAN Chemical), 1 µM AG‐879 (protein Tyrosine Kinase; CAYMAN Chemical), 2 µM IMATINIB (tyrosine kinase; TK1; CAYMAN Chemical), 20 µM CAY10576 (IKKε, IκB kinase episilon; CAYMAN Chemical), 1.5 µM SUNITINIB (multi tyrosine kinase, multi‐TK; CAYMAN Chemical).

Techniques: Isolation, Staining, BrdU Staining, Activity Assay, Derivative Assay