Bz h3c hybrid cell count module

For immunostaining, tissues were fixed with 4% PFA and embedded in paraffin. Sections were incubated with the primary antibody after blocking with goat serum resolved in TBS in Tween 20, followed by incubation with the secondary antibody. Immunostained slides were observed using a fluorescence microscope BZ-X700 (Keyence) and cross-sectional areas of myofibers were analyzed by the BZ-H3C/Hybrid Cell Count Module (Keyence). For ATPase staining, tissues were rapidly frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were incubated with barbital acetate solution at pH 4.11 or 10.43, followed by incubation with ATP incubating solution. For senescence-associated β-galactosidase staining, tissues were fixed in 4% PFA, followed by staining using the corresponding kit (K320-250; BioVision) according to the manufacturer’s instructions. Macroscopic images were acquired with Optio WG-2 (RICOH)^{71 (link)}.

MTT and soft agar assays were performed as described previously (7 (link)). Cells were plated 3000 cells/well in a 96-well plate for the MTT assay. The colorimetric assay was performed with thiazolyl blue tetrazolium bromide. Insoluble formazan was measured by iMark Microplate Absorbance Reader (Bio-Rad) at 595 nm. For the soft agar assay, 500 μl DMEM containing 10% (v/v) FBS, 0.5% (w/v) agarose, and 10 μM TI-4 was plated in one well of a 12-well plate. Then, 500-μl DMEM containing 3000 cells, 10% (v/v) FBS, 0.3% (w/v) agarose, and dimethyl sulfoxide or 10 μM TI-4 was overlaid and kept for 1 h at RT. After that, 250-μl DMEM containing 10% (v/v) FBS was further overlaid and cultured for 2 weeks. To evaluate the effect of the silencing of CSE1L or WWTR1, A549 and U87MG cells were transfected with either control siRNA or WWTR1 siRNA. Twenty-four hours later, the cells were used for the soft agar assay. The images were obtained with a fluorescence microscope BZ-X700 (Keyence) and analyzed by BZ-H3C Hybrid Cell Count Module. The areas of cells were measured from three independent fields.