Fig 2: A B2M-duocarmycin ADC selectively kills senescent cells. (A) Representative Western Blot of B2M expression in EJp53, EJp21, EJp16 and HT1080-p21-9 cells 4 days after induction of p53 or p16 respectively, compared to EJp53 cells transfected with B2M cDNA for 4 days (+ B) . (B) Western blot showing B2M expression for different periods of time (0–6 days) in EJp53 cells transfected with B2M cDNA. (C–D) Cell viability, as measured by Cell Titre Glo, of proliferating and senescent (4 days after induction) EJp53 and EJp16 cells, incubated with different concentrations (C) or only 0.05 µM (bottom) of the B2M ADC or an isotype control for 72 h. An EJp53 cell transfected with B2M for 4 days was used as a positive control (+ B). Line and bar charts show the mean of 4 independent experiments. Colour codes match all panels. Bar represent the mean ± SD. * p < 0.05, ** p < 0.005. (E) Cell death, as measured by PI staining, in the same cells as above. Graphs show percentage of PI positive cells (dead cells). Bars represent the mean ± SD. * p < 0.05, ** p < 0.005.

Fig 3: B2M expression increases with age in mice. (A) Representative images of immunohistochemical staining (left) of skin and brain (frontal lobe) samples from mice of different ages (3–87 weeks), showing B2M expression, as compared to p16 (senescence marker) Magnification: × 20. Graphs show quantitation of expression as %DAB (right). Bars represent the average of three independent samples for skin and five for brain, error bars represent standard deviation (SD). Fold change quantifications of B2M protein (B) and mRNA (C) expression levels, as measured by Western blot and quantitative real-time PCR, respectively, in EJp53, EJp21, EJp16 and HT1080-p21-9 cells induced to senesce for 4 days, normalized to the expression of their proliferating counterparts (represented by the dotted line at y = 1). Bars represent the mean ± SD of 3 independent experiments. ** p < 0.005 (compared to proliferating controls).