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. 2020 Dec 19;45:101149. doi: 10.1016/j.molmet.2020.101149

Figure 6.

Figure 6

Deletion of ERα impaired 7,8-DHF-stimulated activation of BDNF/TrkB signaling cascades. (A) Inhibition of ERα by ICI182780 suppressed 7,8-DHF- or BDNF-induced TrkB activation. Differentiated C2C12 myotubes were preincubated with ICI182780 (5 μmol/L) or vehicle for 6 h and then continued for various time intervals as indicated in the presence of vehicle, BDNF (100 ng/mL), or 7,8-DHF (1 μmol/L). Cell lysates were then collected and the phosphorylation of TrkB (second panel), Src (fourth panel), ERK (sixth panel), and AKT (eighth panel) was examined by immunoblotting. Total ERα (first panel), TrkB (third panel), Src (fifth panel), ERK (seventh panel), AKT (ninth panel), and the expression of UCP1 (tenth panel) and tubulin (eleventh panel) were also verified. (B)–(G) The densitometric measurements of protein bands in (A) and the expression levels of target proteins in the control group (con, without ICI182780, abbreviated as ICI) were normalized to 1. Representative images and densitometries from three independent experiments are shown (n = 3, N.S: not significant, ∗p < 0.05, ∗∗p < 0.01, Student's t test; N.S: not significant, &&p < 0.01, &&&p < 0.001, vs the control group without ICI182780, one-way ANOVA). (H)–(J) ERα gene (Esr1) expressions (H), ERα protein levels (I), and densitometric measurements of ERα protein levels relative to tubulin expression (J). Esr1-KD achieved by lentiviral delivery of short hairpin RNA (one control and three clones tested and marked as Esr1-con, Esr1-KD1, Esr1-KD2, and Esr1-KD3). Densitometric analyses are expressed as means ± SD in arbitrary units normalized to 1.0. n = 5–6 for Esr1 qRT-PCR test in (H); n = 3 for ERα immunoblotting in (I) and (J). ∗∗p < 0.01, ∗∗∗p < 0.001 vs control group, one-way ANOVA). (K) Esr1-KD impaired 7,8-DHF-stimulated BDNF/TrkB-signaling cascades in C2C12 myotubes. Differentiated control (Esr1-con) or Esr1-KD2 C2C12 myotubes were incubated in BDNF (100 ng/mL) or 7,8-DHF (1 μmol/L) for various time intervals as indicated. Cell lysates were then collected and the phosphorylation of ERα at the S118 (second panel) and Y537 (third panel) sites, TrkB (fourth panel), Src (sixth panel), ERK (eighth panel), and AKT (tenth panel) was examined. Total ERα (first panel), TrkB (fifth panel), Src (seventh panel), ERK (ninth panel), AKT (eleventh panel), and the expression of UCP1 (twelfth panel) and tubulin (thirteenth panel) were also verified. Control is abbreviated as con.