Fig. 2: Characterization of GAC reporter at cellular levels.

Relative mRNA levels of Glb1 and mCherry (a) and representative western blots showing the corresponding protein levels (b) in lung tissues from indicated mice. WT, Glb1^+/+ mice (15 months, female, n = 3); HE, Glb1^+/m heterozygous mice (15 months, female, n = 3); HO, Glb1^m/m homozygous mice (15 months, female, n = 3). c Linear correlation between the mRNA levels of Glb1 and mCherry in passages 1 to 5 of Glb1^+/m MEF. d Representative images showing elevated mCherry fluorescence signal in Glb1^+/m MEFs at P7. Arrows indicate senescent cells. e Representative images showing increased SAβ-gal staining (left) and mCherry protein levels (right) in Glb1^+/m MEFs at P5. Arrows indicate senescent cells. f Representative images showing the co-existence of mCherry signal and anti p16^Ink4a fluorescence staining (green) in Glb1^+/m MEFs during replicative senescence. Relative fluorescence staining intensity was quantified in over 100 cells per group. g FACS sorting strategy of mCherry^Low and mCherry^High Glb1^+/m MEFs. h Immunoblotting analysis of mCherry, GLB1 and indicated senescence markers in Young (P1), mCherry^Low and mCherry^High Glb1^+/m MEFs. i qPCR analysis of expression levels of mCherry, Glb1 and indicated senescence marker genes in Young and mCherry^High Glb1^+/m MEFs from n = 3 embryos. Immunostaining of p16^Ink4a (j), p21^Wif1 (k) and Lamin B1 (l) in Young and mCherry^High Glb1^+/m MEFs from n = 3 embryos. Relative staining intensity was calculated from over 85 cells per group. m SAβ-gal staining in Young and mCherry^High Glb1^+/m MEFs from n = 3 embryos. Percent SAβ-gal-positive cells and relative cell size was quantified. Over 100 cells per group were counted. Cell passage (P) numbers are indicated. “n” represents number of biological replicates. Data represent the means ± s.e.m. p value was calculated by Student’s t test (two-sided).