Figure 3. MAP3K4 regulates HDAC6 expression and activity during normal TS cell differentiation and EMT through direct protein-protein interaction.

(A–C) HDAC6 expression and activity are increased upon differentiation of TS cells. Cells were either undifferentiated (0) or differentiated for four days (4) in medium lacking FGF4, heparin, and MEF-CM.

(A) qRT-PCR data show the mean ± SEM of three independent experiments.

(B) Western blots of whole cell lysates are representative of three independent experiments.

(C) Cytoplasmic HDAC activity is expressed as the mean ± SEM of four independent experiments.

(D) Co-expression of HA-MAP3K4 and FL-HDAC6 reduces cytoplasmic FL-HDAC6 activity. FL-HDAC6 was immunoprecipitated from transfected 293 cells and HDAC6 activity and expression were measured. Activity data are the mean ± range and blots are representative of two independent experiments.

(E) FL-HDAC6 was co-expressed with either wild-type or kinase-inactive HA-MAP3K4. FL-HDAC6 was immunoprecipitated and activity measured as described in (D). Activity data show the mean ± SEM of three independent experiments, relative to empty vector. Blots are representative of three independent experiments.

(F–H) The kinase domain of MAP3K4 binds HDAC6 independent of MAP3K4 kinase activity. 293 cells were transfected with the indicated constructs and blots were probed with the indicated antibodies.

(I,J) Endogenous MAP3K4 and HDAC6 co-immunoprecipitate in murine TS cells (I) and human 293 cells (J).

(F–J) Blots are representative of three independent experiments. (FL, Flag; KD, Kinase Domain; KI, Kinase Inactive; WT, wild-type; C, control; sh2, HDAC6 knockdown shRNA2).

Student’s t-test, *p-value < 0.05;***p-value < 0.001.