Upright fluorescence microscope

Female SCID^® mice (5–6 weeks old) were purchased from the Animal Resources Center (Canning Vale, Australia). Mice were injected subcutaneously into the lower rear flank with MDA-MB-231 breast cancer cells (2×10⁶ cells/0.1 mL Matrigel^®). When tumors reached an average volume of ~100 mm³, mice were randomly divided into three groups. The free DiI (15 mg/kg solubilized in 1% DMSO) and SMA-DiI (15 mg/kg equivalent, solubilized in D/W) were administered through oral gavage, while the third group received SMA-DiI (15 mg/kg solubilized in distilled water [D/W]) intravenously. Animals were euthanized in a CO₂ chamber 8 hours posttreatment. For histological examination, all organs were processed as described above. The sections were mounted on a glass slide, stained with Hematoxylin, and representative images were obtained using the Nikon upright fluorescence microscope. The amount of DiI distributed in each tissue was calculated based on the fluorescence intensity across ten sections of the tissue, subtracting the background autofluorescence and normalized based on the area of tissue under observation, using ImageJ software.
All in vivo procedures were approved by the Animal Ethics Committee of University of Otago.

For immunofluorescence analyzes, cells were plated on glass slides in 6-well plates. After 24 h, different concentrations of Ganetespib (STA-9090) or control (vehicle) were added to cells for 72h. Cells were fixed with 4% paraformaldehyde. The fixed cells were incubated with 3% BSA for 30 min and then overnight with the MYC antibody (1:50) at 4°C. After washing (x3) with PBS, the cells were incubated with goat-anti-rabbit antibody (1:50) for 50 min and mounted using fluorescent mounting medium (Beyotime, China). Cells were examined using a Nikon upright fluorescence microscope (NIKON, Japan) and at least 1000 cells from 10 to 15 viewing fields per group were used to calculate percentages of cells.

The cells at logarithmic growth phase were seeded onto the gelatin pre-coated coverslips in the 6-well plates. After 72 h transfection of siPHGDH#4 and siPHGDH#5, cells were then fixed with 4% para-formaldehyde for 20 min, blocked in 2% BSA/PBS for 30 min, and incubated in primary anti-γH2AX antibody (Millipore) overnight at 4 °C. Cells were washed three times in 2% BSA/ PBS, incubated in Alexa Fluor 594 secondary antibody (Invitrogen) for 1 h at room temperature. DAPI was added to stain nuclei. Cells were imaged on an upright fluorescence microscope (Nikon, Melville, NY).

Hybridization was performed overnight with hsa-circ-0046600 probes, sections were washed, and DAPI stain was added and incubated for 8 min in the dark. The sections were observed under a Nikon upright fluorescence microscope, and images were acquired. The nuclei stained with DAPI appeared blue under UV excitation and contained hsa-circ-0046600, which was positively labelled with Cy3 (red emission). The sequence of the hsa-circ-0046600 probe used for FISH was 5ʹ-TTTAG CGTAT CCGAC TGGGT TAGGA GCTGC-3ʹ.