Vectashield solution

The hybridization mix was prepared as follows. In a microtube, 5 ng of the P. jirovecii-specific FISH probe PJ1 (5′-GGCTTCATGCCAACAGTCC-3′, conjugated to a Cy5 fluorophore) and of the previously described Cy3-conjugated pan-eucaryotic probe [37 (link)] each were added to 10 µL hybridization buffer containing 30% formamide as optimized previously. In detail, the hybridization buffer composition was as follows: 0.9 M NaCl, 0.01% w/v SDS, 20 mM Tris-HCl pH 7.2, 30% formamide, and 1 µM of probe. For the FISH reaction, the slides were incubated for 2 h at 55 °C in line with the probes’ calculated melting temperature (see “Results” chapter for details). Afterwards, excess probe was removed by immersing the slides for 20 min in washing solution (5 mM Tris, 15 mM NaCl and 0.1% Triton X-100) at 55 °C as described [20 (link)]. Subsequently, the slides were counterstained with DAPI (4′6′-diamidino-2-phenylindol), which is specific for nucleic acids. After that, the excess dye was removed again in a washing solution, and finally, the slides were air-dried at room temperature and mounted on a glass slide. In detail, the slides were mounted with Vectashield solution (Vector, Burlingame, CA, USA) and examined with a Zeiss Axioskop 40 microscope (Zeiss, Jena, Germany) equipped with a standard filter set as detailed elsewhere [39 (link)].

Immunofluorescence staining was performed in cells cultured on chamber slides. The cells were washed with PBS and then fixed with 4% paraformaldehyde for 10 min at room temperature. The samples were permeabilized with 0.2% Triton X-100 for 5 min and then blocked with 10% bovine serum albumin in PBS for 30 min. Antibodies against Myc and p53 in 1% blocking solution were added to the sample and incubated overnight (4 °C). After washing the samples with PBS 3 times, fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies were added to a 1% blocking solution, and the mixtures were incubated for 1 h. The stained samples were mounted using 4',6-diamidino-2-phenylindole-containing Vectashield solution (Vector Laboratories Inc., Burlingame, CA, USA) to counterstain the nuclei. After 5 additional 5 min washes, the samples were examined using a Leica DM 14000B confocal microscope (Leica, Wetzlar, Germany).

After the irradiation of 2 Gy using X-RAD 320 (Precision X-Ray, North Branford, CT, USA), 2000 cells were plated on 4-well slide plates. After plating, the cells were treated with PI3K kinase inhibitors (GDC0941, GDC0032, CAL101 and IPI145) for 12 hours. The cells were fixed by a 4% paraformaldehyde solution. The cells were permeabilized by incubating in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 for 10 min followed by washing three times with PBS for 5 min. Next, the cells were blocked with 1% BSA in PBS with 0.1% Tween 20 (PBST) for 1 h. The cells were incubated overnight at 4°C with anti-phospho-γ-H2AX antibody (05636I, Millipore, Burlington, Massachusetts, USA), and washed three times for 5 minutes with PBST. The cells were incubated with a fluorescein isothiocyanate-labeled secondary antibody for 1 h in the dark and mounted with VECTASHIELD solution (94 010, Vector Laboratories, Burlingame USA). Observation phospho-γ-H2AX foci were observed and fluorescence images were acquired using the Zeiss LSM 700 confocal fluorescence microscope.

Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, then permeabilized in 0.05% Triton X-100 in PBS and incubated with blocking solution (2% glycine, 2% BSA, 5% FBS, 50 mM NH₄Cl in PBS pH 7.4) for 1 h at room temperature (RT). After incubation with appropriate primary (4 °C) and secondary antibodies (RT), coverslips were mounted with Vectashield solution (Vector, Burlingame, CA, USA). Images were acquired with a Zeiss Axiovert 10 epifluorescence microscope furnished with 40× and 100× Zeiss Apoplan oil immersion objectives and equipped with a Retiga-SRV camera operated with the standard QC capture software (Q-Imaging). Quantification was performed with ImageJ software (NIH, USA) using the corrected total cell fluorescence (CTCF) method, CTCF = integrated density – (area of selected cell × mean fluorescence of background readings), where CTCF is the corrected total cell fluorescence. Both the endogenous and recombinant protein levels were quantified, and the data were normalized to arbitrary units (A.U.).

Bacteria were stained using CFSE (Invitrogen) according to the manufacturer's protocol. J774A.1 cells were seeded on 2-chamber plates and infected with the stained bacteria at an M.O.I. of 10 for 4 or 24 h. Infected cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min and blocked with 1% BSA for 30 min at room temperature. The cells were labelled with rat anti-LAMP1 antibody (#25245; Abcam) or anti-8-oxyhydrodioxy guanosine (8-OHdG-Alexa Fluor 647, sc-393871; Santa Cruz Biotechnology), which is a marker of DNA oxidative damage, incubated for 24 h at 4°C and then labelled with goat anti-rat IgG (H+L) Alexa Fluor 633 conjugate at a dilution of 1: 1,000 for 45 min at room temperature. Nuclei were stained with 300 nM DAPI (#D1306, Invitrogen) for 5 min at room temperature. The cover slips were washed thoroughly with PBS and mounted on glass slides with Vectashield solution (Vector Laboratories) and visualized by fluorescence microscopy using a confocal laser scanning microscope system (Olympus-FV3000).