Author response image 1. Physiological (3%) oxygen condition is inappropriate for MEFs culture to assess the replicative lifespan-extending effect of CQ.

(a) MEFs at passage 1 were cultured in 20% O₂ (black) or 3% O₂ (grey) with treatment of 1 μM CQ (circles) or DMSO (squares), and cell number was determined at each passage. ***P < 0.01. (b) Quantitative RT-PCR analysis of mRNA levels of p16 and p21 genes in different passages of MEFs with or without treatment of CQ in 20% O₂ or 3% O₂. ***P < 0.001. ‘ns’ indicates no significant difference. (c) Representative images showing morphology of MEFs under the indicated culture conditions with or without CQ treatment. (d) Quantification of SA-β-Gal-positive cells under indicated culture conditions. Data represent the means ± SEM. ***P < 0.001; ‘ns’ indicates no significant difference.