Journal: Biomedicines
Article Title: Senescent Macrophages Release Inflammatory Cytokines and RNA-Loaded Extracellular Vesicles to Circumvent Fibroblast Senescence
doi: 10.3390/biomedicines12051089
Figure Lengend Snippet: Raf hyperactivation induces cellular senescence in RAW 264.7 murine macrophages. ( A ) Growth curve of RAW 264.7 cells expressing ∆Raf-1:ER or ER treated every 48 h with vehicle (ethanol) or 4-hydroxytamoxifen (4-OHT, 100 nM). Relative growth was assessed by crystal violet retention assay. Each point represents the mean of a technical triplicate and error bars correspond to the standard deviation. The experiment was performed 3 times (the graph corresponds to one representative biological replicate). ( B ) Western blots showing levels of ∆Raf-1:ER (with ER-α antibody), phospho-ERK, and phospho-histone H3. Protein extracts were obtained from RAW 264.7 cells expressing ∆Raf-1:ER treated as in (A). Tubulin was used as a loading control. The experiment was performed 3 times (the figure corresponds to one representative biological replicate). ( C , D ) RT-QPCR of markers that are upregulated in senescence ( C ) and downregulated in senescence ( D ). RT-QPCR was performed on total RNA extract from RAW 264.7 cells expressing ∆Raf-1:ER and treated for 3 days with 4-OHT or vehicle, as in ( A ). RNA levels were normalized over Tbp and β-Actin. The experiment was performed 3 times. Error bars represent standard deviation; * p -value < 0.05; ** p -value < 0.01; *** p -value < 0.001 using Student’s t -test. ( E ) Senescence-associated ß-Galactosidase staining of RAW 264.7 cells expressing ∆Raf-1:ER treated as in ( C , D ). ( F ) Immunofluorescence (IF) staining of Lamin B1 with DAPI staining performed on RAW 264.7 cells, as in ( C , D ). ( G ) Quantification of IF from F showing the percentage of cells with Lamin B1 alterations. ( H ) Immunofluorescence (IF) staining of γ-H2AX, 53BP-1, and DAPI staining performed on RAW 264.7 cells expressing ∆Raf-1:ER and treated as in ( C , D ). ( I ) Quantification of IF from H showing number of cells with DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). For all IF, experiments were performed 3 times; **** p -value < 0.0001 using Mann–Whitney U test.
Article Snippet: Primary antibodies used for immunofluorescence were phospho-Ser 139 H2A.X (1:100, clone JBW-301, cat #05-636 Millipore), 53BP-1 (1:200, clone Ab-1, at #PC712 Calbiochem, San Diego, CA, USA), Lamin B1 (1:450, cat #ab16048 Abcam, Cambridge, UK), PML (1:300, produced by our laboratory against the peptide comprising amino acids 352–356 of human PML-IV, a region common to all PML isoforms) [ ].
Techniques: Expressing, Standard Deviation, Western Blot, Quantitative RT-PCR, Staining, Immunofluorescence, MANN-WHITNEY
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