Confocal microscopy

Immunostaining was performed using specific antibodies (Supplementary Table S4) as previously described^{50 (link)}. To detect SGs, cells were treated with arsenite (100 μM; Merck) for 30 min before immunostaining. Cells were counterstained with Hoechst33342, washed five times with PBS, mounted on slides, and imaged by confocal microscopy (Carl Zeiss, Oberkochen, Germany and Olympus, Tokyo, Japan).

Animal protocols were approved by the Institutional Animal Care and Use Committee of Northeastern University and carried out in accordance with the approved guidelines. For biodistribution, fluorescence whole-animal and ex vivo tissue imaging was used to monitor the Cy5.5-labeled brush polymer and the Cy5-labeled RNA component of the pacRNAs. To establish an SKOV3 tumor model, 2.0 × 10⁶ cells suspended in PBS were inoculated in female BALB/c nude mice by subcutaneous injection. When the tumor volume reached approximately 200 to 300 mm³, the mice were administered with samples and controls at equal RNA concentrations (500 nmol/kg; free brush concentration equals that of the pacRNA; n = 4) via tail vein injection and scanned at 1, 4, 8 and 24 hours using an IVIS Lumina II imaging system (Caliper Life Sciences Inc. MA, USA). After 24 hours, mice were euthanized using CO₂, and major organs and the tumor were removed for biodistribution analysis. The amount of sample retention in the tissue was normalized to the weight of the corresponding tissues. For the analysis of tumor penetration depth, tumors were immediately frozen in optimal cutting temperature compound (Fisher Scientific Inc., USA) 24 hours after injection. The frozen tumor tissues were cut into 8-μm-thick sections using a cryostat, stained with DAPI, and imaged by confocal microscopy (Carl Zeiss Ltd., Cambridge, UK).

To examine the intracellular localization of the BO extract, we monitored the photosensitizer using confocal microscopy (Carl Zeiss, Oberkochen, Germany). Cells were grown on cover slides and incubated in DMEM containing 40 μg/mL of the BO extract for 1, 2, 4, and 8 h. After incubation, the cells were treated using 4% formaldehyde fixative solution (T&I Co., Ltd., Gwangju, Korea) for 5 min, washed twice with PBS, and then mounted onto slides. Finally, the cells were examined by confocal microscopy at excitation and emission wavelengths of 530–560 and 590–650 nm, respectively.

Cells viability of treated and control cells were tested by DAPI staining. Pretreated HeLa and HCT116 cells with Cp, 10 wt%SPIONs/S-16-A-Cp, 10 wt%SPIONs/S-16-APAA-Cp or vehicle control were washed with 1x PBS then fixed with ice cold Methanol for 5 minutes then stained with DAPI (Sigma Aldrich) for 10 minutes in the dark. Cells were washed again with 1x PBS. The wells were dried up and mounted with ProLong ® Gold Antifade reagent (Thermo Fisher, Waltham, USA). The images were developed by ZEN software using confocal microscopy (Zeiss, Oberkochen, Germany).

SKBR3 cells were grown on poly D-Lysine coated coverslips and treated with vehicle alone, 50 μM NS1643 or 80nM PMA and 2μM Ionomycin in RPMI 1640 media. Following treatment, the cells were washed with 1X PBS, fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton X-100 for 15 min at room temperature. Nonspecific binding was blocked with 5% bovine serum albumin (BSA) for 1h at room temperature and then incubated with 1:100 primary rabbit monoclonal anti-NFAT-1 antibody (Cell signaling Technology, Danvers, MA) diluted in 1% BSA at 4°C, overnight, while cells incubated with 1% BSA alone, served as negative controls. Following incubation with the primary antibody, the cells were washed with 1X PBS and incubated with Alexa fluor 594 conjugated anti-rabbit secondary antibody (Life technoloiges, Carlsbad, CA). Following washes, cells were incubated with 50ng/mL DAPI to stain the nuclei. The coverslips were mounted on VECTASHIELD reagent (Vector Laboratories, Burlingame, CA) and fluorescent images were taken using confocal microscopy (Carl Zeiss Meditec, Inc., Thornwood, NY). Nuclear translocation of NFAT-1 across groups was analyzed and quantified using the analyze particle tool from Image J.

To determine the subcellular localization of the protein CpMAX1a, the ORFs of CpMAX1a without its stop codon were cloned into the pCAMBIA1300 vector by using the SacI and BamHI sites. The obtained plasmid 35S:CpMAX1a-GFP or an empty vector was then introduced. Protoplasts were transformed using the Arabidopsis Protoplast Preparation and Transformation Kit (Coolaber, Beijing, China), according to the manufacturer’s instructions, and the GFP signal observed by confocal microscopy (Zeiss, Germany). Primers used for the plasmid construction are listed in Table S2.