Figure 3. Reversible Diapause-like State Can Be Induced In Vitro by Starvation or Inhibition of mTOR.

(A) Generation of Lkb1 short only isoform. A schematic of Talen-mediated gene editing of the endogenous Lkb1 locus using donor construct that expresses Lkb1-short form-specific exon 9A (Figure 1F) fused directly in frame with exon 8 followed by 2A peptide. This modified Lkb1 locus expresses only short form of Lkb1.

(B and C) Pharmacological inhibition of mTOR and starvation can induce a reversible diapause-like state. (B) mTOR inhibition by INK-128, is reversible as indicated by the rephosphorylation of mTOR, S6 and 4EBP1 24 h after removal of INK-128 in R1(LL) and R1(SS) cells. Beta-actin 1: pmTOR, pS6 and p4EBP1, beta-actin 2: mTOR, S6 and 4EBP1. (C). Starvation abolishes phosphorylation of mTOR and its substrates, S6 kinase, S6, ULK1, and 4EBP1 in 24 h. This inhibition is reversible as indicated by the rephosphorylation of those proteins 24 h after removal of starvation media from culture both in R1(LL) and R1(SS) cells. Intriguingly, the mTOR signal in the untreated and the reversed R1(SS) samples is reduced compared to their respective R1(LL) samples. Beta-actin 1: pmTOR, pS6 and p4EBP1, beta-actin 2: mTOR, S6 kinase, S6 and 4EBP1, beta-actin 3: pS6 kinase, beta-actin 4: pULK1, beta-actin 5: H4K16ac.

(D) Quantification of pmTOR protein bands from C using ImageJ software. n = 3 for each sample, p = 0.0027 for R1(LL) versus R1(SS) 24h after starvation removal. (E) Western blot of pAMPK in mESCs expressing different forms of Lkb1 splice variants. The phosphorylation of AMPK is increased in response to 2DG both in R1(LL) and R1(SS). However, higher levels of AMPK phosphorylation are observed in R1(SS) even in the absence of 2-DG.

(F) A schematic representation of the two splice forms of Lkb1.