Fig 1: Osteopontin is a critical regulator of adipose tissue and macrophage senescence.(A) Representative SA-ß-Gal staining of VAT from 12-month-old WT and OPN–/– mice (selected from n = 5/group). (B) Immunoblot of VAT from 12-month-old WT and OPN–/– mice. Separate loading controls (ß-actin) are presented due to the noncontemporaneous run of the same lysates. (C) Representative immunofluorescence of VAT (arrowheads: p16, p53, and p21). (D) Percentage of p16+ cells (n = 5/group, same data from the aged WT as in Figure 1). Representative immunofluorescence (E) and quantification (F) of p16+ in the percentage of CD68+ cells in VAT (n = 4/group). FACS gating strategy (G) and quantification (H) of p16+ in CD11b+ F4/80+ ATMs from VAT in indicated groups (n = 4/group). (I) Representative images and quantification (n = 5 mice/group) of p16 in VAT of 12-month-old WT treated with AA vs. vehicle (VEH); arrowheads: p16+ cells. (J) Representative images and quantification of p16+ in the percentage of CD68+ in VAT (n = 7–10 mice/group). Arrowheads: double-positive ATMs. (K) Mean fluorescent intensity ratio (MFIR) of OPN receptor expression in CD11b+ F4/80+ ATMs from VAT of 3- and 12-month-old WT mice (n = 3–7 mice/group). (L) OPN receptor expression in CD11b+ F4/80+ ATMs from BMDMs of 3- and 12-month-old WT mice (n = 6–7 mice/group). SA-ß-Gal (M) and p16 (N) staining in BMDMs (from 3-month-old WT mice) treated with recombined OPN protein or VEH ± CD44 blocking antibody (n = 8–9/condition). (O) Protocol of in vivo implantation of Matrigel enriched with OPN protein or VEH. (P) Representative immunofluorescence of excised Matrigel after 14 days of implantation (selected from n = 3 mice/group). Quantification of relative CD68+ area (Q) and percentage of p16+ cells (R) (n = 3 mice/group). All scale bars: 50 µm. Data are presented as original images (A–C, E, G, I, J, M, N, and P) or individual values with mean ± SEM and analyzed with 2-tailed, unpaired Student’s t test (D, F, I–L, Q, and R) or 1-way ANOVA with Tukey’s post hoc test (F, H, M, and N); ns, nonsignificant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.