Dfc350fx

Stock suspensions of LPs, either filled with free-CF640R or empty, were diluted 1:400 v/v in 0.5% low gelling temperature agarose in Ames’ medium at 37 °C. A thin film was polymerized over a pure agarose meniscus in a Petri dish and covered with Ames’ medium. 3D image stacks were acquired with a 63x/0.9NA water immersion objective and a CCD camera (DFC350 FX, Leica Microsystems, Milan, Italy) in an upright widefield fluorescence microscope (DM LFSA, Leica Microsystems, Milan, Italy) using a Cy5 filterset (49,006; Chroma, Olching, Germany). Stacks were deconvolved and max projected along the z-axis using Fiji/ImageJ as detailed in Ref [68 (link)].

Fluorescence images for correlative microscopy were recorded on a Leica DM6000B microscope (Leica Microsystems GmbH, Wetzlar, Germany), equipped with a CCD-camera (DFC350FX) and the following filter cubes.A4 (UV): Exc 360/40, Dichro 400, Suppres BP 470/40. GFP (blue): Exc 470/40, Dichro 500, Suppres BP 525/50. SFRED (red): Exc HQ 630/20x, Dichro Q649LP, Suppres HQ 667/30. Furthermore, this microscope allows imaging in brightfield and phase contrast microscopy.

To assess cell proliferation, MD-ASC and ASC at the third passage in culture were plated at a density of 3.0 × 10³ cells/cm² in a 96-well plate. The medium was replaced with a fresh one every 4 days. Every 24 h for 7 days cells were stained with 1 µg/mL Hoechst 33,342, incubated for 15 min at 37 °C, and images were acquired using an epifluorescence Leica DMI 6000B microscope connected to a Leica DFC350FX camera (Leica Microsystems, Wetzlar, Germany). Cells at each time point were counted using ImageJ 1.52i software. Population doubling time (PDT) was calculated during exponential growth (log phase) with the following formula: PDT = T ln2/ln(Xe/Xb), where T is the time interval considered, Xb is the cell number at the beginning of the time interval, and Xe is the cell number at the end of the time interval. All experiments were performed in triplicate.

The D17 cell line, known to overexpress PLK‐1 and c‐Myc proteins, was selected for inhibition experiments using BI 2536 (Boehringer Ingelheim, Ingelheim, Germany).³² A 10 mM stock solution of BI 2536 was prepared by resuspending the compound in dimethyl sulfoxide (DMSO). D17 cells treated with DMSO were used as control. First, 3 × 10⁵ cells/well were seeded in six wells cell culture plates and were then treated with BI 2536 at 2.5, 5, 7.5, and 15 nM for 12 and 24 h. Morphological changes were evaluated with a Leica AF6000 LX (Leica Microsystems, Wetzlar, Germany) microscope equipped with a Leica DFC350FX digital camera controlled by the LAS AF software (Leica Microsystems). Based on morphological effect, viability assay was performed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) on 1 × 10⁶ cells/well at 2.5, 5, 7.5, and 15 nM for 12 and 24 h. Similarly, caspase activity for apoptosis detection was evaluated using Caspase‐Glo 3/7 Assay System (Promega, Madison, WI) according to the datasheet after 12 and 24 h of treatment at 2.5, 5, and 7.5 nM. The untreated D17 cell line was used as control while the medium was used as blank to subtract background signal. All experiments were performed in triplicate and repeated three times.