Deltavision deconvolution microscope

Before immunofluorescence imaging, SW480 cells were treated with 6 μm CHIR99021(Cat#04-0004; Stemgent) for 24 h in IMEM medium containing 10% FBS. For immunostaining assay, cells were extracted in CSK buffer (100 mM NaCl, 300 mM sucrose, 1 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 10 mM PIPES pH 6.8) plus 0.1% Triton X-100 for 5 min as described previously (Jamieson et al, 2011 (link)). Then, the cells were fixed in 4% paraformaldehyde for 10 min at RT and permeabilized with 0.5% Triton X-100 in PBS on ice for 10 min. After three washes in PBS, cells were blocked in 3% bovine serum albumin for 15 min at RT and incubated with LEF1 (CST 2230s; C12A5) and β-catenin (ab22656) antibodies in a blocking buffer at 4°C overnight. After three washes in PBS, cells were incubated in fluorescent secondary antibodies in a blocking buffer for 1 h. Next, cells were incubated with Hoechst dye for 5 min at RT after three washes in PBS. After three PBS washes, the slides were mounted with the mounting agent (Cat# ZLI-9556) and coverslips were sealed and stored at 4°C. Images were acquired using DeltaVision Deconvolution Microscope (GE Healthcare) with 60× oil immersion objective lens. Image deconvolution and analysis were operated by SoftWoRx software. Image analysis was performed using ImageJ software (National Institutes of Health).

To monitor the expression level and subcellular localization, WT and MmSVCT1 mutants containing a C-terminal GFP followed by a Strep tag were transfected into HEK293T cells (ATCC, #CRL-3216). The cells were maintained in DMEM (Cytiva) with 10% fetal bovine serum (HyClone) and 100 units/ml penicillin plus 100 µg/ml streptomycin. For western blot, cells were harvested 48 h after transfection and solubilized in a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 1% DDM for 1 h at 4 °C. After centrifugation at 20,000 × g for 30 min, equal amounts of samples were resolved on an SDS-polyacrylamide gel and transblotted onto a PVDF membrane. The membrane was then probed with 1:2000 diluted mouse anti-StrepII-tag monoclonal antibody (ABclonal, AE066, Clone No. AMC0521) and 1:5000 diluted goat HRP-conjugated anti-mouse IgG (BBI Life Sciences, D110087).
Immunofluorescence imaging was performed following the published protocol^{56 (link)}. HEK293T cells were grown on coverslips and transfected with MmSVCT1 variants. After 24 h, the cells were fixed with 3.7% formaldehyde in PBS buffer. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) from Sigma. Images were collected via the FITC channel (for the GFP signal) or DAPI channel on a DeltaVision deconvolution microscope (GE Healthcare). Data were captured and processed using DeltaVision softWoRx software V6.5.2 (Applied Precision).

Rhizopus nigricans and Aspergillus flavus were obtained from the clinical microbiology laboratory and maintained on yeast extract peptone dextrose (Life Technologies, Grand Island, NY, USA) agar. The isolates were anonymous with respect to patient origin and did not derive from any of the autopsies. Both species were cultured in yeast extract peptone dextrose broth for 8–10 h at 37 °C, washed in tris-buffered saline, pH 7.4 and then heat-killed at 80 °C for 2 h in a water bath and washed in tris-buffered saline, pH 7.4 with 4% bovine serum albumin and 2 mM Ca⁺⁺. Fungi were fixed on microscope slides and incubated with human serum (the source of SAP) or tris-buffered saline, pH 7.4, with 4% bovine serum albumin and 2 mM Ca⁺⁺ for 2 h, washed with buffer and incubated with SAP antibody (Biocare Medical) for 1 h. The slides were washed again with the same buffer and fungi incubated with 1/500 Alexa Fluor 555 goat anti-rabbit IgG (Life Technologies) in tris-buffered saline with 4% bovine serum albumin and 2 mM Ca⁺⁺ for 1 h, washed and observed with a DeltaVision deconvolution microscope (GE Healthcare, Issaquah, WA, USA). All incubations were performed at 26 °C. Both antibody preparations were pre-absorbed against the respective heat-killed fungi overnight at 26 °C.

For RNA FISH, 5 days post-transduction, CRISPRa and Tet-on KILR overexpressing cells grown on coverslips were fixed in 4% formaldehyde for 10 min followed by permeabilization in 70% ethanol overnight at 4 °C. Cells were then stained for 16 h with 125 nM of a custom KILR Stellaris RNA-FISH probe set labelled with Quasar 570 fluorophore (LGC Biosearch Technologies) according to the manufacturer’s instructions. For IF, CRISPRa, CRISPR-Cas13, Tet-On and RPA1-KILR overexpressing cells were challenged with or without 6 Gy gamma irradiation followed by 6 h of incubation. The cells were then treated with CSK buffer (10 mM PIPES, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1.4% Triton X-100) to remove the cytoplasm, followed by fixation in 4% formaldehyde for 10 min and permeabilization with 0.5% Triton X-100 for 15 min. The cells were incubated with antibodies against RPA1/RPA70 (Abcam, ab79398, 1:250), γH2AX (Abcam, ab2893, 1:1000) or RAD51 (GeneTex, GTX70230, 1:500). Coverslips were mounted onto slides using ProLong Glass antifade medium containing NucBlue nuclear counterstain (Thermo Fisher Scientific). Images were acquired with the DeltaVision Deconvolution microscope (GE Healthcare) using a 60x objective lens and analyzed with ImageJ software. A minimum of 100 cells per sample were analyzed.

HCT116 cells were cultured to 70% confluency in a 60-mm dish. KaryoMAX^TM Colcemid^TM Solution in PBS (Gibco, #15212012) was added to a final concentration of 0.02 μg/ml together with DMSO (control) or 1 µM 5-Ph-IAA. Treated cells were incubated at 37 °C 5% CO₂ for 2 h before trypsinization. Removed cells were treated with 75 mM KCl before fixation in MeOH/acetic acid (3:1) fixative solution. Fixed cells were adjusted to approximately 10⁷ cells/ml. 10 μl of the cell suspension was applied onto a glass slide and dried at room temperature. 10 μl of DAPI-containing Vectashield Mounting Medium (Vector Laboratories, #H-1200) was added before sealing with a coverslip. Chromosomes were observed under a DeltaVision deconvolution microscope (GE Healthcare).