Staurosporine

HUVECs were cultured in 12 well plates (Thermofischer Scientific, USA) at a density of 4 × 10⁴ cells/well and incubated in Endothelial Cell Growth Medium with antibiotics, glutamine, and heparin (PELO Biotech) supplemented with 2.5% FBS. 17-19 h later, the cells were washed and incubated in medium with 4 mM H₂O₂ (Merck, Darmstadt, Germany), 200 nM staurosporine (Abcam), 2 µg/mL HrA2 and vehicle for 2 and 24h. The morphology of HUVECs was observed under an inverted phase contrast microscope (microscope: AXIO Vert.A1, Zeiss, camera: AxioCam ICc1).

Apoptosis was determined using Fluorescein Isothiocyanate-Conjugated (FITC) Annexin V and PI kit (Thermos Fisher Scientific, Eugene, OR). Normal live cells have Phosphatidylserine (PS) positioned on the inner leaflet of the plasma membrane. One of the hallmarks of early apoptosis is the translocation of PS to the outer leaflet of the plasma membrane. The exposed PS becomes available to bind Annexin V, a protein that binds PS with high affinity [14 (link)]. PI stains dead cells, as previously mentioned. Annexin V positive cells emit green fluorescence (excitation/emission: 488/518 nm). To induce apoptosis, cells were treated with 1 μM staurosporine, a protein kinase inhibitor (Abcam, Waltham, MA), and incubated for 4 hours which served as a positive control sample. According to the manufacturer’s recommended protocol, Annexin V/PI was added to cells and incubated in the dark for 15 minutes, then analyzed immediately with a flow cytometer (Bigfoot2 Cell Sorter, Invitrogen, Waltham, MA). 10,000 events were collected for each cell sample. The flow cytometry data was subsequently analyzed using FCS express 7 software to compare apoptotic fractions in control and needle ejected cells.

Recombinant mouse GluOC was prepared as described^{8 (link)}. Myristoylated PKI 14-22 amide (PKA inhibitor) was obtained from Enzo Life Sciences (Farmingdale, NY), U0126 (MEK inhibitor) and AS1842856 (FoxO1 inhibitor) from Merck-Millipore (Darmstadt, Germany), garcinol (p300 inhibitor) and necrostatin-1 (RIP1 inhibitor) from Abcam, staurosporine (inducer of apoptosis) and carvacrol (TRPM7 inhibitor) from Wako (Osaka, Japan), and neutralizing antibodies to FasL from R&D Systems (Minneapolis, MN).

Bacteria were added to triplicate wells of HeLa cell monolayers for infection as described in LDH assay. Caco2 cells were plated at 2 × 10⁵ cells/ml in 24 wells. All infections were carried out at a MOI of 10. Gentamicin was added at 100 µg/mL to each well after 2 hr of infection to kill extracellular bacteria. At each indicated time point, monolayers of HeLa cells were washed with PBS and cells were lysed by incubation with PBS containing 0.5% Triton X-100 for 10 min at room temperature with agitation. Serial dilutions of lysates were plated on MMM plates and incubated at 30°C overnight for subsequent CFU enumeration. Z-VAD-FMK (EMD Millipore) was added to HeLa cells at the beginning of infection at 50 µM where indicated. Z-VAD-FMK at this concentration prevented apoptotic cell-death induced by 1.2 μM staurosporine (Abcam). When needed HeLa cells were treated with either 100 µM Cholesterol (Sigma) along with a carrier, randomly methylated β-cyclodextrin (MCD) (Cyclodextrin Technologies Inc) to increase membrane Cholesterol levels or 1% (w/v) hydroxypropyl β-cyclodextrin (HPCD) (Cyclodextrin Technologies Inc) to deplete Cholesterol along with gentamicin treatment.

To induce PS translocation, BMMs and primary osteoblasts were seeded at a density of 10 × 4 cells/cm², cultured, and differentiated for 48 h and 7 days, respectively. Cells were harvested by trypsinization, washed first in growth medium and then washed in PBS (HyClone, Logan, UT, USA) supplemented with 0.5 mM CaCl₂. Subsequently, the cells were treated with 10 mM ionomycin (Abcam, Cambridge, UK) or ethanol for 5 min or 2.5 μM staurosporine (Abcam, Cambridge, UK) for 8 h at 37 °C. ionomycin was removed by washing in ice-cold PBS. Treated cells were then washed in annexin-binding buffer and stained with Alexa Fluor^® 488 annexin V and propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA) according to standard operating procedures. Gating strategy: (1) implement FSC/SSC gate for living cells, (2) define the gate for FITC- or FITC + cells, (3) apply this gate to all samples. Flow cytometric analysis was conducted using a BD LSR Fortessa (Becton Dickinson, Heidelberg, Germany), and the data were processed with FlowJo v.10.7.