Extended Data Figure 2: Autophagy contributes to ADAR1 downregulation during senescence.
a-b, Expression of the indicated proteins was determined by immunoblot in IMR90 cells treated with vehicle control or etoposide to induce senescence with or without simultaneous treatment of Lys05 (a) and relative ADAR1 levels were quantified (b). The experiment was repeated three times independently with similar results.
c-d, Expression of the indicated protein was determined by immunoblot in proliferating (PD28) and replicative senescent (PD73) IMR90 cells treated with or without Leupeptin (c) and relative ADAR1 levels were quantified (d). The experiment was repeated three times independently with similar results.
e, Expression of the indicated proteins was determined by immunoblot in young and proliferative IMR90 (PD30) treated with or without Lys05 (e). The experiment was repeated three times independently with similar results.
f-g, Expression of the indicated proteins was determined by immunoblot in IMR90 cells induced to undergo senescence by oncogenic H-RASG12V with or without expressing shATG7 (f) and relative ADAR1 levels were quantified (g).
h-k, Expression of the indicated proteins was determined by immunoblot in control early passage proliferating or late passage replicative senescent IMR90 (h) or WI38 cells (j) with or without ATG7 knockdown. And relative ADAR1 levels were quantified in i and k.
l-m, Representative confocal images of immunostaining of endogenous ADAR1 and LC3 in control proliferating and RAS-induced senescent IMR90 (l). And the percentage of cells with co-localization of cytoplasmic ADAR1 puncta and LC3 (as exemplified by arrows pointed puncta) were quantified in 4 independent biological repeats (m).
n, Co-immunoprecipitation analysis between ADAR1 and LC3 in cytoplasmic extracts from the indicated proliferating (PD21) and replicative senescent (PD68) IMR90 cells. Downregulation of ADAR1 was validated in whole cell lysates.
o, IMR90 cells transfected with mCherry-ADAR1 p150 were induced senescence by expressing oncogenic H-RASG12V. Localization of mCherry-ADAR1p150 was visualized and DAPI counter staining was used to visualize nuclei. Scale bars = 10 μm.
Data represent the mean ± s.e.m. of three biologically independent experiments unless otherwise stated. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.