Figure 3. ZAKβ responds to cellular compression but not stretching.

• A
  Schematic of mechanical perturbation of cells by compression and stretching.
• B
  U2OS and ΔZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.
• C
  U2OS and ΔZAK cells stably expressing the indicated Strep‐HA‐tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).
• D
  U2OS and ΔZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).
• E
  U2OS and ΔZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).
• F
  Model of MAP3Ks involved in p38‐ and JNK‐activating signaling in mechanically stimulated cells. ZAKβ is activated upon compressive load (left), in a manner dependent on stress fiber binding. TAK1 is activated by cell stretching (right).
• G
  Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM—transcripts per million.
• H
  Murine C2C12 myoblasts can be differentiated into myotubes by incubation in a myotube differentiation (diff.) medium for 6 days.
• I
  C2C12 cells were incubated in diff. Medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.
• J
  C2C12 myoblasts and myotubes (6 d differentiation) were pretreated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.

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