4 6 diamidino 2 phenylindole (dapi)

HnRNPU protein expression alteration in NCl-H226 cells was observed by immunofluorescence 48h after cells were transfected with SFTA1P overexpressing vector. After growing for 12h, cells transiently transfected with the PCDNA3.1-SFTA1P or empty vectors were inoculated in special petri dish. After transfected for 48h, the cells were fixed with 4% paraformaldehyde, then washed in PBS at room temperature 3 times. Then the cells were permeabilized with 0.1% Triton X-100 (Beyotime) in PBS for 30min at room temperature, then washed in PBS three times. After incubated for 30min, 1% BSA incubated in PBST was used to block the binding of non-specific sites. 60 min later, primary antibody of hnRNPU (1:100, ab10297) in 1% BSA was added and the cells were incubated for 60min at room temperature. Then 20μl fluorescence labeling antibody (1:50, Beyotime) was added and incubated for 30min. Nuclei were stained with DAPI (Cat. No A1001, Applichem, Germany) at a concentration of 10 ng/ml in cold dark place for 15 min. Finally, cells were observed by fluorescent microscope.

Image acquisition and analysis were performed in 96-well plates. Cells were fixed using 3,7% formaldehyde, and cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (AppliChem, A1001). Cells were kept in PBS, and images were acquired using the IN Cell Analyzer 1000 (GE Healthcare) using the Nikon Plan Fluor ELWD 40 × 0,6 objective. In each well of a 96-well plate, 20 images were taken, each with an average of 10 cells per image. Subsequent automated image analysis was performed using IN Cell Analyzer Workstation 3.4 software^{9 (link),30 (link)}.

Regarding cell cycle assay, 100 μl of cell suspension (harvested as for apoptosis assay) were incubated with 4’,6-diamidino-2-phenylindole (DAPI) solution (AppliChem, #A4099) and measured using FACS Canto II flow cytometry 280 system. Data were analyzed with the software FlowJo 10.1r1 (FlowJo LLC). All further calculations and statistical analyses were performed using Graph Pad Prism 7.0 software.

Immunofluorescence staining was performed using standard protocols14 (link). Cells grown on coverslips or in collagen gels were fixed in 4% paraformaldehyde and permeabilized with 0.2% TritonX-100. After washing cells were blocked with 1% BSA and stained as per experimental requirement with Alexa Fluor 488 Phalloidin (A12379, 1:40, Invitrogen), SMAD3 (51-1500, 1:250, Invitrogen) and DAPI (A1001,0010, 1 μg/μl, Applichem) for nuclear staining. Mounting was done in fluorescent mounting medium (S3023, DAKO). Fluorescence micrographs were captured using Q-capturePro (QImaging, Surrey, Canada), a fluorescence imaging software driving a Retiga CCD camera (QImaging, Surrey, Canada) mounted on an Olympus IX81 microscope (Olympus Europa Holding GmbH, Hamburg, Germany).

In 4 mammosomatotropinomas, confocal laser scanning microscopy (Olympus FV1000D, Japan) was performed using the same primary antibodies (GH/NeuroD1 and PRL/NeuroD1 cocktail). Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 647 goat anti-mouse (Abcam, UK) were used as secondary antibodies. Nuclei were stained with DAPI (appliChem). Details of the confocal laser scanning microscopy method are given in the Supplementary Materials.

The physical accumulation of the complexes were assessed by using Cy5-labeled pDNA (Mirus, USA). Equal amounts of labelled pDNA were administered, regardless of the N/P ratio of the complexes (e.g. N/P4 included 20 μg of labelled pDNA, whereas in case of N/P2, 20 μg of labelled pDNA was mixed with 30 μg of unlabeled pDNA). For the histological evaluation, the lungs were inflated postmortem with 1:1 cryomatrix:PBS (O.C.T embedding matrix, Kaltek, Italy), frozen in 2-methylbutane bath on dry ice. Fresh cryo-sections (10 μm thick) were fixed with ice cold methanol, counterstained with DAPI (AppliChem) and imaged with confocal microscopy (Zeiss LSM710).
The data are presented as the mean +− standard deviation.