Research Paper Volume 9, Issue 8 pp 1867—1884

p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

Figure 1. Induction of p16Ink4a and SAβG in macrophages does not require p53. Peritoneal lavage and alginate beads containing SCs (AB) were recovered from wild type (p53+/+) or p53 knockout (p53-/-) mice 15 days after injection (AB model). (A) Representative microphotographs of cryosectioned immunocyte capsules surrounding alginate beads stained with May-Grünwald-Giemsa for histology (10x objective), X-Gal substrate for β-galactosidase activity (SAβG; pH 6.0) (blue) with nuclear fast red counterstain (red), and an immunofluorescent antibody against macrophage marker F4/80 (red) with DAPI nuclear counterstain (blue) (40x objective). (B) Total yield of cells recovered from peritoneal lavage from naïve or AB-injected p53+/+ and p53-/- mouse strains. (C) AB model-elicited immunocyte capsules were pooled equally from 3 mice and p16Ink4a gene expression relative to internal reference gene β2-microglobulin (B2m) was measured by qPCR. (D) β-galactosidase activity from cell extracts of immunocyte capsules from alginate beads recovered from individual mice was measured via 4-MUG hydrolysis, presented as the rate of 4-MU fluorescence (RFU) per minute normalized per microgram of protein. (E) Representative microphotograph of adherence-selected peritoneal lavage from naïve and AB-injected mice stained with X-Gal for SAβG activity. Data show mean ± standard deviation of two independent experiments (n=3 mice per experiment). Statistical comparison between p53+/+ and p53-/- strains are indicated; ns, not significant; **, p-value < 0.01.