Bafilomycin

The chemical compounds rapamycin (Merck KGaA, Darmstadt, Germany) and bafilomycin (Thermo Fisher Scientific, Waltham, MA, USA; Cat# PHZ1164) were used. Before drug administration, a hole was made in the chorion of 8-hpf embryos, whereas 24-hpf embryos were dechorionated. All drugs were dissolved in DMSO and stored in small aliquots at −20 °C. We performed 1 µM rapamycin treatment from 8- to 48-hpf and 40 nM bafilomycin treatment for 16 h prior to the end of the experiments, specifically either from 32- to 48-hpf or 16 h before 8-dpf. Starvation was induced starting from 6-dpf for two days. After treatments, embryos/larvae were collected in Eppendorfs, anesthetized, and vials were placed on ice. After 3 washes with cold PBS, embryos/larvae were deyolked in PBS with the addition of PMSF and centrifuged for 5 min at 4 °C at 5000 rpm. After centrifugation, supernatants were discarded, and samples were stored at −80 °C.

LPS from E. coli 026:B6 (Sigma-Aldrich) and ATP (Invivogen) were used at concentration of 50 ng/ml and 3 mM for human and 100 ng/ml and 5 mM for murine co-culture studies, respectively. Nigericin (Invivogen) was used at a concentration of 10 µM. To evaluate autophagy response, WT and WAS KO BMDCs or WT and WAS KO THP-1 cells were stimulated with rapamycin (50 nM; Calbiochem) and bafilomycin (160 nM; Thermo Fisher Scientific) alone or in combination. All murine IFNs were from Millipore and human cytokines from Peprotech. BMDCs were stimulated with 500 U/ml IFN for 16 h prior to LPS priming (100 ng/ml) for 3 h, followed by ATP (5 mM) for 30 min.

Murine MCs were harvested by peritoneal lavage as follows: naive mice were sacrificed by cervical dislocation and 5 mL of lavage buffer (2 mM EDTA, 0.25% BSA in PBS) was injected into the peritoneal cavity and incubated for 3 min prior to collection. MCs were identified within the harvested cell suspension as described in the flow cytometry section using a LSR Fortessa flow cytometer (BD Biosciences) and FlowJo software (TreeStar) and subsequent parameters were analyzed. To quantify MC senescence, cells were incubated in suspension in OptiMEM buffer (ThermoFisher Scientific) containing 100 nM of Bafilomycin (ThermoFisher Scientific) for 1 h at 37°C in order to increase the intracellular pH. The cell suspensions were then supplemented with 33 μM of C₁₂FDG (ThermoFischer Scientific) for 2 h prior to analysis of C₁₂FDG MFI. MC apoptosis was assessed as previously described (Asai et al., 2001 (link)). Briefly, peritoneal cells were stained with fluorescently labeled primary mAbs as described in the flow cytometry section and incubated with 500 μl of Annexin V binding buffer (140 mM NaCl, 2.5 mM CaCl₂ and 10 mM HEPES at pH 7.4) containing 5 μl of FITC-Annexin V (BD) and 5 μl of propidium iodide (Biolegend) for 15 min. Apoptotic cells were defined as Annexin-V⁺/propidium iodide^-. MC granularity was assessed using the flow cytometric side scatter profile.

Endogenous JAM-C or CBL was depleted in HUVECs by 2 rounds of transfection with 250 pmol siRNA as above. At the second round, cells were transfected with WT or Quad-K JAM-C–GFPout and plated on borosilicate glass-bottomed dishes (Greiner Bio One, Kremsmunster, Austria). The following day, cells were imaged in the presence or absence of 100 nM bafilomycin (Life Technologies) in a heat-controlled chamber at 37°C with 5% CO₂ in HGM using either a 63× oil immersion objective (NA 1.3) and a Zeiss 800 microscope (Zeiss, Jena, Germany) or, for higher speed images, a 100× oil immersion lens (NA 1.4) and a spinning disk (UltraVIEW VoX; Perkin-Elmer, Waltham, MA, USA). During longer-term time lapses (45 min), images were acquired every 45 s at a resolution of 512 × 512 pixels and a step size of 0.5 μm. During spinning-disk acquisition, images were acquired every 5 s for 10 min with a step size of 0.4–0.5 μm, comprising 9–14 pictures (depending on cell height) with an exposure for each image at 30 ms.