Supplementary Materialsmolecules-24-04445-s001. and modulation of EMT elements, as well as increased expression of phospho-H2A.X, support further pre-/clinical investigations, including the analyses of these markers. 0.05, t-test (b) Representative images of the PANC-1 scratch areas in control (untreated cells) and in cells treated with CX-5461 over time. 2.3. CX-5461 Induces mRNA Expression of EMT Markers The migratory capability of cells is often linked to an altered expression of epithelial to mesenchymal transition (EMT) or mesenchymal to epithelial transition (MET) markers. Therefore, we investigated the effect of CX-5461 on the mRNA expression levels of snail (SNAI1), slug (SNAI2), E-cadherin (CDH1), N-Cadherin (CDH12), vimentin (VIM), and matrix metalloproteinase 9 (MMP9). The cells were exposed to CX-5461 for 24 h. Interestingly, we observed slightly different effects in the different cellular models. For instance, SNAI1 and SNAI2 were increased in SUIT-2-28 and PDAC-3 (Figure 3a,b), whereas SNAI1 and MMP9 remained unaltered in PANC-1 after drug exposure (Figure 3c). Moreover, CX-5461 treatment increased the expression of CDH1 in PDAC-3 and PANC-1 cells, whereas no alteration was observed in SUIT-2-28. These results suggest that CX-5461 hampers the cells in the epithelial phenotype, but further studies should investigate other aspects underlying the relative MPH1 contributions of inhibition of Pol I in these PDAC cells considering the effects on migration versus EMT. Open in a separate window Figure 3 Effect of CX-5461 on the mRNA expression of EMT markers. (a) SUIT-2-28 (b) PDAC-3 (c) PANC-1. Significance * SRB in 1% acetic acid), and resuspension in 10 mM Tris buffer, the optical density was measured at 490 and 540 nm on a BioTek plate reader (BioTek Instruments Inc., Winooski, VT, USA). 4.4. Migration Assay PANC-1 cells were seeded in 96-well plates with a density of 30,000 cells per well to form a confluent monolayer overnight. Subsequently, the cells were scraped with a 96-well pin tool scratcher and detached cells were removed by washing steps TBB of phosphate-buffered saline (PBS). Medium only or medium containing 1.5 M CX-5461 was added to the wells and brightfield images were taken with software Universal Grab 6.3 digital (Digital Cell Imaging Labs, Keerbergen, Belgium) on a Leica DMI300B microscope (Leica Microsystems, Eindhoven, The Netherlands) at various time points. The obtained images were analyzed using the Scratch Assay 6.2 software (Digital Cell imaging Labs, Keerbergen, Belgium). 4.5. qRT-PCR Cells were treated with 1.5 M CX-5461 or TBB medium for 24 h and RNA was isolated according to the TRIzol reagent protocol (15596-026, ThermoFisher Scientific, Waltham, MA, USA). One microgram of RNA was then used for cDNA synthesis and subsequent PCR using the First-Strand cDNA synthesis kit (K1612, ThermoFisher Scientific, (Waltham, MA, USA). 4.6. Immunofluoresent Staining and Imaging Cells were seeded on VWR 18 18 mm cover glasses (thickness 1.5) and incubated for 24 h for attachment. Subsequently, the cells were treated either with drug-free medium or medium containing 1.5 M CX-5461 for 24 h. After three washing steps with PBS, cells were fixed with 200 L 4% paraformaldehyde (PFA) (15710, Electron Microscopy Sciences, Hatfield, PA, USA) diluted in PBS for 10 min at room temperature (RT). After three other washing steps, cells were permeabilized 10 min at RT with 0.1% Triton x-100 (108643, Merck, Amsterdam, The Netherlands) diluted in PBS prior to overnight incubation at 4 C with primary antibody Phospho-H2A.X (#2577, cell signaling, 1:50). Secondary antibody incubation was performed with Abberior STAR 488 (ST488, Abberior, G?ttingen, Germany) and actin (TRITC conjugated phalloidin, Sigma-Aldrich, Zwijndrecht, The Netherlands) for 2 h at RT, followed by 15 min DAPI incubation. The coverslips were mounted in PBS and images were obtained on a widefield Zeiss Observer Z1 microscope (Zeiss, Jena, Germany) equipped with a CCD camera (EXI Aqua, QImaging, Surrey, BC, Canada). Illumination of samples was performed by TBB an HXP 120 C lamp (Zeiss, Jena, Germany), and light was collected using a 63X oil immersion objective (NA = 1.4, Zeiss, Jena, Germany). The following filters were used: FITC (475/40 and 530/50?nm for excitation and emission filters, respectively), DAPI (365 and 445/50 nm for excitation and emission filters, respectively), and TRITC (545/25 and 605/70 nm for excitation and emission filters, respectively). Image processing was performed using ImageJ (NIH). 4.7. Western Blot Whole-cell lysates were prepared from cells treated with 1.5 M CX-5461 for 24 h or medium as control, by addition of cell lysis buffer (#9803, Cell signaling, Leiden, The Netherlands) diluted in demineralized water and supplemented.