Plectin-deficiency results in decreased keratin network branch length but does not alter overall cellular stiffness and cytoplasmic viscoelasticity.

<p>A431 cells (wild type) and AK13-1 subclones stably expressing HK13-EGFP and either scramble control shRNAs or plectin shRNAs were grown for 48–57 h on laminin 332-rich matrices. <b>A</b> Histogram of branch length-measurements. Confocal fluorescence images of the bottom plane of isolated single cells were recorded to measure the HK13-EGFP filament length between branch points by automated image analysis. Results from either scramble control shRNA clones 1 and 2 or plectin shRNA clones 1 and 2 were combined. The plectin-deficient cells have significantly increased branch length in the absence of FCS which is even more apparent in the presence of FCS. The whiskers are min to max and statistical differences were determined by two-tailed Mann-Whitney test. <b>B</b> 3D confocal microscopy (maximum intensity projections are shown) of live cells after 48 h growth in the presence of 10% FCS. Note that mesh size and bundling of the keratin filament network is increased in the plectin-deficient cell. <b>C</b> Histogram depicting apparent stiffness A_1.8 of single cells that was determined from indentation experiments that were performed above the nucleus in plectin shRNA clone 1 and scramble control clone 1. Error bars are standard deviation. <b>D</b> Graph showing the average forces needed to reach different indentation depths above the nucleus (same cells as in C). <b>E</b> Creep compliance measurements of magnetic tweezers experiments. The rescaled peaks [n_shPlectin = 117 (20 cells), n_control = 107 (19 cells), n_{wild type} = 67 (12 cells)] were fitted to equation 1 and averaged for each phenotype. Error bars are standard deviation. Note that no significant differences in viscoelastic properties of the cytoplasm were observed.