Fig 2: eIF2A affects lipid accumulation in primary adipocytes. (A) Standard primary adipocytes isolation and differentiation protocol were followed, and cells were stained with Oil Red O (ORO) at day 4 post-differentiation. (A) Primary preadipocytes isolated from White Adipose Tissue (WAT) of ~8-week-old male mice and induced for differentiation. Microscopy of undifferentiated, differentiated and ORO-stained cells is shown on the left. Quantitation of the staining results (absorbance at 500 nm is shown on the right; average of the three independent experiments utilizing tissues from 3 different mice). (B) Primary preadipocytes isolated from WAT of ~8-week-old female mice and induced for differentiation. Microscopy of undifferentiated, differentiated and ORO-stained cells (on the left). Quantitation of the staining results (absorbance at 500 nm is shown; average of the three independent experiments utilizing tissues from 3 different mice) (on the right). (C) Primary preadipocytes isolated from brown adipose tissue (BAT) of ~8-week-old male mice and induced for differentiation. Microscopy of undifferentiated, differentiated and ORO-stained cells (on the left). Quantitation of the staining results (absorbance at 500 nm is shown on the right; average of the 3 independent experiments utilizing tissues from 3 different mice). (D) Primary preadipocytes isolated from BAT of ~8 weeks old female mice and induced for differentiation. Microscopy of undifferentiated, differentiated and ORO-stained cells (on the left). Quantitation of the staining results (absorbance at 500 nm is shown; average of the 3 independent experiments utilizing tissues from 3 different mice) (on the right). *p < .01 (Student's t-test)

Fig 3: Survival of mice of the indicated genotypes and genders and assessment of behavior and locomotor activity. (A) Comparison of longevity group survival between WT (C57BL/6J) mice and eIF2A-KO mice (males and females monitored over 1 year) (Number of animals used for the analysis: WT Males = 83, eIF2A-KO Males = 65, WT Females = 85, eIF2A-KO Females = 84). (B) WT and eIF2A-KO female mice were placed in individual home cages, and their locomotion was assessed every hour for 72 h (with 12-h light and 12-h night [shaded grey] periods) (n = 3 for each group). (C) WT and eIF2A-KO female mice locomotion was assessed under constant darkness every hour for 72 h (n = 3 for each group)

Fig 4: eIF2A affects the expression levels of autophagic receptor, p62, in primary adipocytes upon differentiation. (A) Western blot of key adipogenic and autophagic markers: PPAR?, LC3B (represented by LC3-I (upper band) and LC3-II (lower band) isoforms) and p62 in wild-type and eIF2A-KO adipocytes isolated from WAT and BAT tissues before and after differentiation. (B) Real-time PCR. Relative C t values are shown; C t values were normalized to the actin signal and plotted relative to undifferentiated p62 mRNA levels. All data presented as means ± SEM, N = 5 independent points per each RT-PCR

Fig 5: eIF2A affects adipokine expression in the liver and adipose tissue. (A) Analysis of adipokine expression in mice liver tissues. (B) Analysis of adipokine expression in mice WAT and BAT tissues. Mouse adipokine array with captured detection antibodies was incubated with tissue lysates followed by the chemiluminescent detection. Data analyses were done using the HLImage++ Array analysis software from Western Vision. Averages of the 3 independent experiments utilizing tissues from 3 different mice for liver tissue samples and from 4 different mice for adipose tissue samples are shown. Tissue samples were prepared as outlined in the materials and methods section. Two-tailed Student's t-test was used for calculating the p-values and statistical analysis of the data. p-values: *< .05; **<. 01