Luciferase

Changes in extracellular ATP level were detected with a luciferin/luciferase-based chemiluminescence assay emitting green light. Perfusion of brain slices was halted and 50 μl aliquots containing 12 mg/ml luciferin (Sigma) and 5 mg/ml luciferase (Sigma) were added to a 1 ml bath chamber filled with aCSF at room temperature (because luciferase is not stable at higher temperatures). Calcium was uncaged and imaged from single astrocytes as described above, using the red Ca²⁺ indicator Rhod-2 (50 μM) instead of Fluo-4. A field of view containing an astrocyte and its processes was selected and ATP-derived bioluminescence was collected in darkness using highly sensitive GaAsP detectors with a Zeiss LSM780 2-photon/confocal microscope.

Protein extracts were lysed using 2× SDS lysis buffer (50 mM Tris, pH 6.8, 20% glycerol, 2% SDS), and 2× SDS loading buffer (100 mM Tris, pH 6.8, 20% glycerol, 4% SDS, 0.04% bromophenol blue) was added. Extracts were run on an 8% SDS-PAGE gel, transferred to Immobilon-P transfer membranes (Millipore Corp., Bedford, MA), and visualized with antibodies specific for IKKα (Cell Signaling), IKKβ (Cell Signaling), IκBα (Santa Cruz), phospho-IκBα (Cell Signaling), IE86 (monoclonal antibody [MAb] 810; Millipore), luciferase (Sigma), and GAPDH (Abcam). The relative intensity of bands detected by Western blotting was quantitated using ImageJ software.

At 24 hours after surgery, mice (n = 8/group) were re-anesthetized and the integrity of the vascular barrier was quantified as we have previously described [37 (link)]. Each mouse was injected with luciferase (Sigma, St. Louis, MO) through the jugular vein (100 μl/30 g body weight). At 25 minutes after the injection, 20 μl of venous blood was obtained and diluted in 80 μl of PBS. At 30 minutes after the injection, each animal was intracardially perfused with PBS and the cord was extracted. Both the cord and blood sample were quickly frozen on dry ice.
A 9 mm length of cord, centered over the lesion epicenter, was divided into 3 segments of equal length and weighed. Each segment was then homogenized in cell lysis buffer at a 1:50 dilution. The homogenate was subjected to centrifugation (8 minutes at 12,000 rpm) and 15 μl of supernatant was collected and incubated in 1,400 μl of cell lysis buffer at room temperature for one hour. Then 15 μl of sample was added to 100 μl of substrate from the luciferase Assay System (Promega, Madison, WI) and mixed by pipette. The luminosity of the reaction was measured in a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Values were expressed as percent of the luminosity, as determined in the blood sample.

Acetaminophen (cat # A7085) and diclofenac sodium salt (cat # PHR1144) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The V-7 preservation solution was supplied by Vitron (Tucson, AZ, USA) [32 (link)]. Waymouth’s MB 752/1 (without L-glutamine, phenol red and sodium bicarbonate) culture medium and fetal bovine serum were purchased from Invitrogen (Chicago, IL, USA); while L-glutamine, Antibiotic/Antimycotic solution (100×) and gentamicin sulfate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mixed cellulose-ester IMMOBILIN-NC filters (HATF, 0.45 µm surfactant and triton free, autoclavable), used to support the liver slices on top of the rollers, were obtained from Millipore (Bedford, MA, USA). Dithiobis-nitrobenzoic acid, reduced GSH luciferin, luciferase and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The following antibodies were used in this study. The source of each antibody is indicated. Rabbit::H3K9me3 (Abcam #ab8898 1:4000), LAP2α (Abcam #ab5162 1:500), SIRT1 (Abcam #ab7343 1:200); Rabbit: Mouse: HP1γ (Millipore Sigma #05-690 1:200), Lamin A/C (Abcam #ab40567 1:200), GFP (Invitrogen, #A11122, 1:250); Luciferase (Sigma-Aldrich, #L0159, 1:200); Collagen I (Cedarlane Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200), Laminin (Abcam, #AB11576, 1:1000), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75) and anti-NCAM-Biotin (clone HCD56; BioLegend; #318319, 1:75), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75), and anti-NCAM-Biotin (clone HCD56; BioLegend; #318319, 1:75).

The isolated mitochondrial membrane potential (Δψ) was measured by monitoring the fluorescence spectrum of JC-1 [14 (link)]. The experiments were performed at 37°C in an incubation medium containing 1.5 mL JC-1 staining solution, 2 M pyruvic acid sodium, and 0.8 M malate with 0.3 mg mitochondrial protein. ATP synthase activity was determined using a bioluminescence technique [15 (link)]. The mitochondrial suspensions were added to a cuvette containing 40 μM luciferase (Sigma, MO, USA), 0.25 M sucrose, 3.0 mM Hepes, 0.5 mM EDTA, 2 M pyruvate, and 0.8 M malate. After a background bioluminescence was established for correction, 0.5 μM ADP was added to initiate the reaction. ATP production was monitored at 37°C using a BioOrbit 20/20n luminometer (Turku, Finland) and expressed as nanomoles per minute per mg protein.

Stable cell lines were generated as Flp-In U2OS T-REx cells, or U2OS and MDA MB-435 cells expressing Plk4 (SHCLNG-NM_014264, Sigma), Luciferase (SHC007, Sigma), RFP (Tony Pawson laboratory, Lunenfeld Tanenbaum Research Institute) or FAM46C (SHCLND-NG_017709, Sigma) short hairpin RNAs (shRNAs) through lentiviral infection as described^{19 (link)}. Stable cells transduced with shRNAs were studied within 3 weeks of infection. For transfection in the rescue protocol, 100,000 U2OS FAM46C shRNA (two shRNA constructs utilized) or RFP shRNA cells were seeded onto 6-well plates and transfected after 24 h with 1 μg of RFP-FAM46C or RFP X42h. RFP-FAM46C and RFP expression were confirmed with immunofluorescence. Cells were tested for mycoplasma contamination.