Phalloidin tritc

Immunofluorescent and phalloidin-TRITC staining were carried out on cells fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min [anti-CRIM1 (HPA000556, Sigma) at 1:100; anti-Active-β-Catenin (05–665, Millipore) at 1:1000]. To stain for F-actin, cells were incubated with 50 μg/ml phalloidin-TRITC (Sigma) for 40 min. Imaging was done on Leica DM50000B and Zeiss LSM 510 Meta confocal microscopes. Maximum intensity projections were compiled using ImageJ (NCBI).

Serum starved PC3 or DU145 cells plated on coverslips were treated with PBS, Sema4D-Fc, Sema3C-Fc (R&D, Abingdon, UK, 2 μg/mL), or dexamethasone (Sigma, 10 nM) for 60 min. Cells were then fixed (4% paraformaldehyde), permeabilized (0.2% triton), stained by immunofluorescence with mouse anti-GR(CST) followed by anti-mouse Alexa Fluor488 (Life Technologies, Carlsbad, CA, USA), phalloidin-TRITC (Sigma) and DAPI. Serum starved 22Rv1 cells were treated with PBS, Sema4D (2 μg/mL) or dihydrotestosterone (DHT, Sigma, 1 nM) for 60 min. Cells were then fixed (4% paraformaldehyde), permeabilized (0.2% triton), stained by immunofluorescence with mouse anti-AR followed by anti-rabbit Alexa Fluor488 (Life Technologies), phalloidin-TRITC (Sigma) and DAPI. Images were taken at x63 magnification using a Zeiss LSM510 confocal microscope and the intensity of staining of Alexa488 in the cytoplasm and nucleus was measured using ImageJ using DAPI staining to outline the nucleus and actin staining to identify the cytoplasm. The ratio of nuclear to cytoplasmic staining for each cell was calculated. (n = 3, A minimum of 44 cells were scored per treatment).

The cellular uptake was
assessed by confocal laser scanning microscopy (CLSM) imaging (C 2s,
Nikon). In a typical route, the PC-3 cells were seeded on WillCo glass
dishes (15 × 10³ cells/cm²), incubated
at 37 °C, and treated for 72 h with 250 μg/mL of dye-labeled
L-SSH MNPs. Then, the sample was washed twice with PBS, fixed with
4% paraformaldehyde (PFA, Sigma-Aldrich) for 30 min at 4 °C,
and finally stained with tetramethylrhodamine (TRITC)-phalloidin (100
μM, Millipore) and Hoechst 33342 (1 μg/mL, Invitrogen).
The same procedure was performed for CM-L-SSH, LN1-L-SSH, and CM-LN1-L-SSH
MNPs. Due to the superior targeting ability of CM-LN1-L-SSH MNPs to
target PC-3 cells, the following experiments on magnetothermal stimulation
have been carried out using this sample.
ICP-OES has been performed
to evaluate the intracellular uptake of functionalized MNPs (L-SSH,
CM-L-SSH, LN1-L-SSH, and CM-LN1-L-SSH MNPs). For the quantitative
analysis of functionalized MNP internalization, PC-3 cells were seeded
in T75 flasks (15 × 10³ cells/cm²) for
72 h, washed twice with PBS, detached with 0.05% trypsin-EDTA, and
centrifuged. Then, the samples were digested and measured in a similar
manner used for the ICP-OES analysis of pristine MNPs (SH, SHS, SS,
and SSH MNPs). Finally, data were normalized for the nonfunctionalized
controls (L-SSH MNPs).

The cell area was analyzed for 30 cells for each condition after 24 h of culture using ImageJ (version 1.53e). For cytoskeleton analysis, hUC-MSCs were plated at an initial cell density of 1.5 × 10⁴ cells/well onto the glass coverslips, which were clean (control) or covered by different PEM films. The cells were cultured for 24 h and fixed in 3.7% formaldehyde, solubilized with 0.1% Triton X-100, immunostained with mouse monoclonal anti-human vinculin IgG (Merck, Darmstadt, Germany) and Alexa Fluor 488-conjugated goat anti-mouse IgG-clone A11001 (Merck, Darmstadt, Germany), and counterstained with TRITC-phalloidin (Merck, Germany). Nuclei were stained with DAPI (Merck, Germany). The specimens were mounted onto coverslips with poly(vinyl alcohol) (Dako Fluorescent Mounting Medium, Agilent Technologies, Santa Clara, CA, USA), visualized under a fluorescence inverted microscope (Axio Vert.A1, Zeiss, Dresden, Germany), and analyzed using ZEN 2.3 (Zeiss) software.