Confocal fv1000

Cells were treated with cisplatin (8 µM) and/or Biochanin A (100 µM) for the indicated time points and fixed in 4% paraformaldehyde, followed by incubation with γH2AX antibody (9718 S, CST) at 4 °C overnight. Cells were then incubated with DyLight 594-conjugated secondary antibody for 1 h and stained with DAPI (1 µg/ml) for 3 min. The labeled nuclei were detected by Confocal FV1000 (Olympus). The percentage of cells with ≥ 10 foci was quantified. At least 100 cells were counted per well.

CHO cells were analyzed by fluorescence microscopy using an Olympus Confocal FV1000. The program FV10-ASW 3.0 was used for all acquisitions and settings for the live imaging. For spinning disk acquisition, we used a Quorum Technologies WaveFX Confocal Microscope with a Yokogawa CSU-X1 Spinning Disc Unit and a LCI Temperature Controller and CO₂ Mixer for Live Cell Imaging (Neurobiology of Stress CNS, University of Toronto at Scarborough, Canada). The resulting movies series were corrected to improve the contrast and resolution using the deconvolution plugin on ImageJ. The image analysis of Saf growth and contraction was carried out using ImageJ.

MPC5 cells transfected with the mouse MKL1 expression plasmid or empty vector were seeded onto 24-well plates at 50-60% confluence. Cells were incubated with 50 μM EdU for 2 h at 48–72 h after transfection. After the 2-h pulse, the cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. The cells were subsequently washed with glycine (2 mg/ml) for 5 min, added 0.2% Trion X-100 for 10 min, washed with PBS twice, and added click reaction buffer (Tris–HCl, pH 8.5, 100 mM; CuSO4, 1 mM; Apollo 550 fluorescent azide, 100 mM; ascorbic acid, 100 mM) for 30 min while protecting from light. The cells were then washed again with 0.5% Triton X-100 for three times, stained with Hoechst (5 mg/ml) for 30 min at room temperature, washed with 0.5% Triton X-100 for three times. Images were taken and analyzed using Confocal FV1000 (Olympus). EdU positive cells were calculated with (EdU add-in cells/Hoechst stained cells) × 100%. At least 200 cells were counted per well.

RAW or PC grown in control medium were washed three times with PBS and plated in 24 well-plates. In some experiments cells were incubated in control medium or in the presence of the drugs for 2 h prior to infection with T. cruzi Y-GFP (10:1 parasite:cell ratio) and continued for additional time of 24 h. In other cases, the cells were infected during 24 h and, after washing, treated with drugs for an additional time of 24 h or incubated in control medium during 24 or 48 h before fixation. Cells were then fixed with 3% paraformaldehyde solution in PBS for 15 min at room temperature, washed with PBS, and blocked with 50 mM NH₄Cl in PBS. After washing, cells were mounted with Mowiol containing Hoechst and examined by confocal microscopy, using an Olympus Confocal FV1000 (Japan). Images were processed with the program FV10-ASW 1.7. Confocal images of 10 random fields were quantified, representing around 100 cells per experiment. Data are presented as mean values and error bars indicate the SEM from at least three independent experiments.

Tissue samples were placed in 4% paraformaldehyde in PBS over 2 hours at 4°C, immersed in 30% sucrose overnight at 4°C, and then were embedded in OCT compound (Tissue-Tek, Miles) and sectioned at 5 mm (Leica CM 1850, Leica Instruments) for the morphological and immunohistochemistry study. Sections were washed three times in PBS for five minutes each and incubated in blocking buffer with 10% serum of the secondary antibody host species for 1 hour at room temperature. After that, sections were incubated with rabbit polyclonal Ab against MKL1 (ab49311; Abcam) and p21 (10355-1-AP; Proteintech) at 1:100 dilution overnight at 4°C. After being washed three times for 10 minutes with PBS at room temperature, sections were incubated with Alexa 488-conjugated anti-rabbit IgG for 3 hours at room temperature. Negative control samples were treated with species-appropriate IgG instead of primary antibody. Images were taken and analyzed using Confocal FV1000 (Olympus).