Extended Data Fig. 3: Senescence regulates CD38 in vivo.

a, WT macrophages were incubated with LPS (100 ng/mL) in the presence or absence of 100 nM AP20187 for 20 hours. CD38 activity was measured in cell lysates (n=4 biologically independent samples). b, WT macrophages were treated with LPS (100 ng/mL) for 20 hours and cell lysates were prepared. Lysates were incubated with or without AP20187 for 15 min before CD38 activity was measured (n=3 biologically independent samples). c-g, 12 month-old INK-ATTAC mice were treated with vehicle or with AP20187 for 16 months (28 month-old groups) and were compared with 12 month-old animals. (c) Representative images of immunofluorescent staining for CD38 (red), ORF1 (yellow), and CD45 (green) in WAT. Insets show the image of 28 month-old WAT with removal of green color channel CD45 signal (left) and with removal of red color channel CD38 signal (right) to show accumulation of CD38⁺ CD45⁺ cells near ORF1⁺ cells. Graph shows quantification of CD45⁺ immune clusters in WAT, based on 20 5x fields per sample (12 mo, n=5; 28 mo, n=4; 28 mo+AP, n=3 mice). (d) IL6 levels in WAT detected by ELISA (12 mo n=5; 28 mo n=7; 28 mo+AP n=6 mice). (e) NMN levels measured in WAT (12 mo n=5; 28 mo n=6; 28 mo+AP n=13 mice). (f) Relative mRNA levels of p16 (12 mo n=6; 28 mo n=7; 28 mo+AP n=5 mice) and Cd38 (12 mo n=10; 28 mo n=9; 28 mo+AP n=6 mice) detected by qRT-PCR analysis in liver. Levels are relative to 12 month-old mice. (g) NAD⁺ levels in liver (12 mo n=6; 28 mo n=7; 28 mo+AP n=15 mice). h, Relative mRNA levels of inflammatory and senescence-related genes in X-ray irradiated WT mouse pre-adipocytes determined by qRT-PCR. Levels are relative to control non-senescent cells (Ctrl) (Ctrl n=5; X-ray n=6 biologically independent samples). i, Quantitative analysis of cytokines/chemokines in conditioned media harvested from irradiated (CM-SEN) or non-senescent (CM-NS) mouse pre-adipocytes. Heat maps reflect analytes (pg/mL) measured in 3–25-plex Luminex assays. Heat map shows average of 5 biologically independent samples. Data are mean ± SEM, except letters e-g that are mean ± SD, analyzed by unpaired two-sided t-test.