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. 2014 Jun 29;33(16):1784–1801. doi: 10.15252/embj.201487808

Figure 7. Depletion of MEKK2, MEKK3 or ERK5 inhibits the enhanced differentiation triggered by XIAP depletion.

Figure 7

A HSM cells were transiently co-transfected with siRNAs against XIAP and MEKK2 or ERK5, and differentiation was initiated with the appropriate media the next day. Coverslips were fixed and stained everyday as described (Shown are data from day 6 of differentiation).

B The cells were lysed and the expression of various differentiation markers was tested.

C Pharmacological inhibition of ERK5 prevents the enhanced differentiation provoked by XIAP depletion. HSM were transiently transfected with siXIAP and treated with DMSO or 5 μM of XMD 8–92, and differentiation was started the day after. Transfection was performed every 2 days, and media and drug treatment were renewed every following day. The polynucleation index was calculated as mentioned in (A).

D Experiments were performed as mentioned in (C) and cell lysates were prepared. The expression of various differentiation markers was tested by Western blots.

E HSMs were transiently co-transfected with siRNAs against XIAP and MEKK3 and differentiated as described for (A). Polynucleation of MHC positive cells at day 6 was quantified, and the average fold increase is shown in the right panel (n = 3, Student's t-test, *P < 0.05 and ***P < 0.001).

F, G HSMs were transfected with different siRNAs and differentiated as described in (A). Cells were lysed, run on SDS–PAGE, and blotted for various differentiation markers.

H Schematic model of MEKK2 ubiquitination by XIAP and its effect on the MEKK2/3-MEK5-ERK5 pathway in response to various stimuli. XIAP regulates this cascade by direct interaction and ubiquitination. At steady state, XIAP competes with MEK5 in binding to MEKK2. Upon stimulation with growth factors, XIAP–cIAP1 complex ubiquitinates MEKK2/3, thus leading to the inactivation of ERK5. Loss of XIAP promotes human myogenic differentiation in a MEKK2/3-ERK5-dependent manner. Depending on the stimuli, MEKK2/3 might bind and activate other effectors like MKK7 or p62, thus leading to JNK or NF-κB activation respectively.

Source data are available online for this figure.