Inhibition of MLCK induces apoptosis in vivo. Paraffin-embedded tissue sections of tumors removed from control mice (A to D) and from mice receiving ML-7 (E to H) were stained with FITC-TUNEL (B and F) and DAPI (C and G). Phase-contrast (A and E) and merged TUNEL/DAPI (D and H) images are also shown. Tumors from mice receiving ML-7 (F) showed significantly higher numbers of TUNEL-positive cells than tumors removed from control (B) mice. The cyan color in panels D and H indicates the colocalization of the TUNEL and DAPI staining. Bar = 25 μm.

Destabilizing the cytoskeleton leads to MLC₂₀ dephosphorylation and apoptosis. Treating SMC with 10 μM cytochalasin D (CytD) and growing cells on polyHEMA (poly) overnight resulted in significant decreases in MLC₂₀ phosphorylation (A) and significant increases in cell death (B) compared to the untreated controls (Un). Treating cells with various concentrations of blebbistatin also induced cell death (C) and MLC₂₀ dephosphorylation (inset). *, P value <0.05, t test (n = 3).

MLC₂₀ dephosphorylation during apoptosis. (A) NIH 3T3 cells were treated with 500 nM actinomycin D for the indicated times. MLC₂₀ phosphorylation, caspase activation, and cell death were quantified as described in Materials and Methods. The inset shows the migration of the MLC₂₀ species (un-, mono-, and diphosphorylated MLC₂₀ from top to bottom). Note the initial increase followed by a decrease in MLC₂₀ phosphorylation. *, P value <0.001, ANOVA (n = 3). (B) The stoichiometry of MLC₂₀ phosphorylation (see inset) and cell viability were quantified in untreated SMC (Un) and SMC treated with 10 μM dexamethasone (Dex), 10 μM actinomycin D (ActD), 100 μM cycloheximide (Chx), or 2 μM camptothecin (Cam) for 24 h. *, P value <0.001, t test (n = 3).

Caspase inhibition protects against apoptosis but not MLC₂₀ dephosphorylation. (A) SMC were left untreated or treated with 20 μM ML-7 or 10 ng/ml TNF-α plus 5 μM cycloheximide (CHX) for 24 h. Other SMC were treated with 50 μM z-VAD-fmk for 1 h followed by 20 μM ML-7 or 10 ng/ml TNF-α plus 5 μM cycloheximide for 24 h. z-VAD-fmk significantly increased cell viability compared to cells treated with ML-7 alone, or with TNF-α plus cycloheximide. *, P value <0.01, t test (n = 3). (B) Cells were treated with 50 μM z-VAD-fmk for 1 h, 20 μM ML-7 for 4 h, or 50 μM z-VAD-fmk for 1 h followed by 20 μM ML-7 for 4 h. The cells were then fixed and stained with TUNEL reagent and DAPI. There were very few TUNEL-positive nuclei (blue) in untreated cells or cells treated with z-VAD-fmk. Most nuclei in cells treated with ML-7 were TUNEL positive as indicated by the cyan color from the colocalization of the blue (DAPI) and green (FITC-TUNEL) stains. z-VAD-fmk decreased the number of TUNEL-positive nuclei in ML-7-treated cells.

Inhibiting MLCK leads to MLC₂₀ dephosphorylation and apoptosis. SMC were treated with the indicated concentrations of ML-7 for 16 h (A and B) or with 20 μM ML-7 for different times (C). *, P value <0.001, t test (n = 3). (C, inset) Western blots of cell extracts were probed with antibodies against the inactive 32-kDa caspase-3 precursor or the larger fragment (17 kDa) of activated caspase-3 (top and bottom rows, respectively). Note that the cleaved caspase-3 appears at 8 h following the addition of ML-7. It is preceded by MLC₂₀ dephosphorylation, which takes place within two hours of treatment and before the cells become annexin V positive. *, P value <0.001, ANOVA (n = 3). Panel D shows confocal images of cells microinjected with a control antibody or an affinity-purified inhibitory antibody to MLCK immediately following microinjection (top two rows) and 4 h later (bottom two rows). In this experiment, the control antibody (affinity-purified goat anti-human IgG) was mixed with FITC-labeled dextran prior to injection and the MLCK antibody was mixed with rhodamine-labeled dextran. Although the control antibodies (green cells) had no effect, the cells injected with the MLCK antibody (red cells) displayed morphological characteristics typical of apoptotic cells. These are representative micrographs of cells from three separate experiments.