Figure 3.

MRTF interacts with AurA and inhibits its degradation; non-transcriptional effects of MRTF

(A) LLC-PK1 cells were transfected with the indicated siRNAs, and 24 hr later placed in serum-free medium supplemented with vehicle (DMSO) or the proteasome inhibitor MG-132 (20 μM) as indicated. After an additional 24 hr, cell lysates were prepared for Western blotting and probed for the indicated proteins.

(B) Experiments as in A were quantified by densitometry, and results were normalized to the level of NR-transfected DMSO-treated control is each experiment (n = 3). ∗p < 0.05, ∗∗p < 0.01, the corresponding groups were compared by t test.

(C) LLC-PK1 cells were transiently transfected with plasmids encoding HA-tagged MRTF or Myc-tagged AurA or the combinations of these and 24 hr later HA-MRTF was immunoprecipitated with anti-HA. Protein levels in the input and the precipitates were detected by immunoblot using anti-HA or anti-Myc antibodies.

(D) Endogenous MRTF was immunoprecipitated from LLC-PK1 cells. Total cell lysates and immunoprecipitates were probed with the indicated antibodies.

(E) LLC-PK1 cells were transfected with siNR or siMRTF, and 24 hr later, the level of indicated proteins was examined in the total cell lysates (left) and in immunoprecipitates after pull-down with anti-AurA (right).

(F) Quantification of the various protein-protein interactions. The amount of coprecipitating proteins were normalized to the corresponding amount of immunoprecipitated AurA (n = 3-4, mean ± SD). Results were compared to interactions determined in NR-transfected cells, using ratio t test, ∗p < 0.05, ∗∗p < 0.01.

(G) Differential capacity of wild-type (WT) and TADless (TL) MRTF to exert transcriptional effects. LLC-PK1 cells were co-transfected with the smooth muscle actin (SMA) promoter firefly luciferase construct and renilla TK reporter (for normalization) along with either an empty vector (pcDNA, control) or plasmids encoding HA-tagged WT or TL MRTF. (n = 3, mean ± SD). The results verify that TL MRTF has lost its transactivation capacity.

(H) LLC-PK1 cells were serum-deprived and transfected with the porcine-specific MRTF (psMRTF) siRNA, then re-transfected with siRNA-resistant (murine) HA-tagged wild type (WT) or transcriptionally inactive (TADless, TL) MRTF. Cells were then re-exposed to serum and after 24 hr fixed and co-stained with anti-HA (to visualize MRTF-retransfection) and acetylated tubulin (to visualize the PC). Successful downregulation of MRTF-A and B by psMRTF is shown under the images.

(I) Quantification of cells with cilia in non-transfected (NT) neighbors and in WT- or TL-expressing cells. In each experiment, 25–35 cells were counted; ∗∗p < 0.01. Note that both WT and TL support cilium resorption. Bar: 20 μm.