Modfit lt v5

Cells were synchronized by serum starvation for 72 h prior to treatment with 5 or 10 nM paclitaxel, 0.75 µM SMI#9 or both for 24 or 48 h. Cells were fixed with 70% ethanol, incubated with DNase free RNase A, stained with propidium iodide (PI) and measured using a Northern Lights full spectrum flow cytometer (3-laser configuration; Cytek Biosciences, Fremont, CA) at the Microscopy, Imaging, and Cytometry Resources Core of Karmanos Cancer Institute. An unstained control was used to unmix cellular autofluorescence from PI fluorescence during acquisition using SpectroFlo software (Cytek). Flow Cytometry Standard (FCS) files were exported, then imported into FlowJo (FlowJo LLC, Ashland, OR) where they were gated to remove cell aggregates (based on PI-Height vs PI-Area) and the 2 N DNA peak position was normalized. Gated, normalized FCS files were exported, then imported into ModFit LT v.5.0 (Verity Software House, Topsham, ME) which deconvoluted the DNA histograms using the tetraploid model with debris and apoptosis modeling.

Cell cycle was evaluated by quantifying intracellular DNA using PI (Sigma-Aldrich, St. Louis, MQ, USA) through a flow cytometry-based approach. The exposed cells were trypsinized, washed with PBS, and centrifuged at 2000 rpm at 4 °C for 5 min. Afterwards, the pellets were resuspended in 500 µl PBS and then fixed by drop-wise addition of 2 ml pre-chilled 75% ethanol (cooled at − 20 °C in advance) at 4 °C overnight. The fixed cells were washed twice with PBS and subsequently incubated with PI (50 µg/ml) in the presence of RNase (100 µg/ml) (Sigma-Aldrich, St. Louis, MQ, USA) at 37 °C in the dark for 1 h. Finally, DNA contents of the stained cells were analyzed by a flow cytometer (BD Biosciences, Heidelberg, Germany). The histogram of cell cycle distribution was generated from 10,000 events per sample. The data were eventually presented as percentages of the cells in G0/G1, S, and G2/M phases using the CellQuest software (BD Biosciences, Heidelberg, Germany) and Modfit LT V5.0 (Verity Software House, Topsham, ME, USA).

Two experiments were performed. Cells were seeded into six-well plates in duplicate at the same density as in the DHT-stimulated growth assay. On the next day, hormone-stripped medium supplemented with 0, 1, 10, or 100 nM DHT or fresh K-SFM was changed to the cells. Seventy-two hours after the medium was changed, the cells were trypsinized, fixed, and stained according to the protocol provided by the manufacturer for the propidium iodide staining solution (Invitrogen/Thermo Fisher Scientific). Cells were run with a CytoFlex S flow cytometer (Beckman Coulter, Brea, CA, USA). Unstained samples from the first experiment were used to verify that GFP expressed from the AR vector did not bleed to the PI channel. The cell cycle data were analyzed with ModFit LT v5.0 (Verity Software House) using gating with PI-A/PI-H, followed by manual analysis with ploidy set to diploid and manually defined G1 and G2 peaks. Aggregates and debris were also modeled. Statistical differences in the cell cycle states between conditions were tested with GraphPad Prism v5.02 using one-way ANOVA followed by Tukey’s post hoc test.

MDA-MB-435 cells were synchronized by serum starvation for 96 h, and cells were transfected with NT or MYH9 siRNAs, or treated with 37.5 μM blebbistatin or 25 μM ML7, 8 hr after release into complete media. Control untreated and inhibitor treated cells were collected after 24, 48 or 72 hr. siRNA transfected cells were collected after 48 h and at 96 h after a second round of transfection. Cells were fixed with 70% ethanol, incubated with DNase free RNase A and propidium iodide, quantitated by BD LSR II SORP flow cytometer (BD Biosciences, San Jose, CA, USA) at the Microscopy, Imaging, and Cytometry Resources Core of Karmanos Cancer Institute and analyzed using using ModFit LT v.5.0 (Verity Software House, Topsham, ME, USA).