Intensilight c hgfi

After treatment with different concentrations of TFLS, PCa cells (PC3, DU145) were washed twice with PBS and seeded on coverslips in a 12-well plate overnight. After washing with PBS, the cells were fixed in 4% paraformaldehyde for 30 min. Following fixation, the coverslips with PCa cells were washed with PBS three times and then permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Cells were then washed with PBS three more times before incubating with primary antibodies against NF-κB p65 at 4°C overnight. The next day, the slides were stained with corresponding Alexa Fluor^® 555-conjugated secondary antibodies for 2 h at room temperature. The cell nuclei were then stained with DAPI for 10 min and examined by fluorescence microscopy (Nikon Intensilight C-HGFI, Nikon Corporation, Japan).

Time-lapse wide-field microscopy was performed as previously described (Hung et al., 2011 (link); Albeck et al., 2013 (link)). Briefly, 1000–2500 cells were seeded 2–4 days prior in glass-bottom 24-well (MatTek) or 96-well plates (MGB096-1-2-LG-L; Brooks Life Sciences, Chelmsford, MA), with well bottom pretreated with type I collagen (BD Biosciences, San Jose, CA) to promote cell adherence. For experiments with drug addition, cells were placed in imaging medium until the addition of drug diluted as a 5x-20x concentrated spike. Cells were maintained in 95% air and 5% CO₂ at 37°C in an environmental chamber. Images were collected with a Nikon (Tokyo, Japan) 20×/0.75 NA Plan Apo objective on a Nikon Eclipse Ti inverted microscope, equipped with a Lumencor SOLA or a Nikon Intensilight C-HGFI light source. Fluorescence filters were from Chroma (Bellows Falls, VT): T-Sapphire (89000 ET Sedat Quad; or ET405/20x, T425LPXR, and ET525/50 m), YFP (89002 ET ECFP/EYFP; or 41028), and RFP (49008 ET mCherry; or 41043 HcRed). With a Hamamatsu Photonics (Hamamatsu, Japan) ORCA-ER or ORCA-AG cooled CCD camera or an Andor Zyla 5.5 scMOS camera, images were acquired every 3–8 min with 2 × 2 binning and an exposure time of 200–225 ms for T-Sapphire, 70–225 ms for YFP, 70–225 ms for RFP.

A total of 5.0 × 10⁴ of 3T3 cells per well in a 96-well plate were co-cultured with the 1.0 μg/mL of SA samples for 72 h. The viability of the 3T3 cells was assayed by using 10% CCK-8 reagent for 60 min to determine the toxicity of the SA samples. Afterwards, the cells were fixed with glutaraldehyde, followed by staining with 0.1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for the imaging of the nuclei. An inverted fluorescent optical microscope (Nikon, Eclipse Ti-S, Japan) was utilized. The fluorescent light source was Nikon Intensilight C-HGFI, and two fluorescent filter cubes (Ex = 340–380 nm, Em = 435–485 nm, corresponding to DAPI, and Ex = 465–495 nm, Em = 512–558 nm, corresponding to SA samples) were used to observe the distribution of SA samples in the cytoplasm. A scientific-grade CCD (Dhyana, model: 400 BSI, Tucsen, Fuzhou, China) was used to record the images. The magnification of the OM was 200×. The original fluorescent images were recorded in greyscale. ImageJ software (an open-source software, NIH Image, version 1.54f, Bethesda, MD, USA) was employed to merge and convert two fluorescent images into visible colors, e.g., blue for cell nuclei and green for SA1–SA7 samples.