Fig. 5. Activation of estrogen signaling suppressed autophagy and adipogenesis.

a, b Estradiol (0.1 μM, days 0–12) increased the steady-state protein levels of LC3 and p62 during 3T3L1 preadipocyte differentiation, analyzed by Western blotting (a) and densitometry (b). DI+ represents 3T3L1 cells with differentiation induction and harvested on day 12. c, d The presence of E2 (0.1 μM, days 0–12) reduced autophagy activity during preadipocyte differentiation. On day 12, autophagy flux was analyzed by Western blotting (c) and densitometry (d) after the cells were incubated with and without autophagy inhibitor BL (bafilomycin A1 at 0.1 μM and leupeptin at 10 μg/ml) for 4 h. In densitometric analysis, the band densities of investigated proteins were normalized against that of β-actin, and the fold changes were calculated by taking the normalized density of E2- group as “1”. For autophagy flux analysis, we first normalized the band densities of LC3-II and p62 against that of β-actin, then calculated the differences of normalized densities in the presence vs. the absence of autophagy inhibitor; lastly, the differences were shown as fold changes by taking the E2- group as “1”. e The presence of E2 (0.1 μM, days 0–12) suppressed the differentiation of 3T3L1 preadipocytes, analyzed by Oil Red O staining on day 12. f The presence of autophagy inhibitor bafilomycin A1 (4 nM) and leupeptin (0.4 ng/ml) during days 0–12 recapitulated the effects of E2 on adipogenesis, analyzed by Oil Red O staining on day 12. n = 3–4; *p < 0.05; **p < 0.01; ***p < 0.0001 (with E2 vs. without E2)