All materials were from Sigma unless otherwise indicated. LPS was from E. coli strain 026:B6. Protease inhibitor (PI) cocktail was from Roche. “NONOate” NO donors and 1400W were from Cayman Chemical and freshly prepared in 10 mM NaOH. Sources of antibodies were: α-Txnip/Vdup1 mouse mAb (MBL International, #K0205-3), α-GAPDH mouse mAb (Millipore, #MAB374), α-COX2 rabbit pAb (Cell Signaling, #4842), α-VASP rabbit pAb (Cell Signaling, #3112), α-phospho-VASP rabbit pAb (Cell Signaling, #3111), α-iNOS mouse mAb (BD Biosciences, #610328), α-Flag M2 mouse mAb for immunoblotting (Sigma, #F1804), α-Flag rabbit pAb for immunofluorescence (Cell Signaling, #2368), α-catalase mouse mAb (Sigma, #C0979), α-PCNA mouse mAb (Cell Signaling, #2586).
All cell lines were obtained from ATCC. The RAW264.7 and HEK293 cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin. For transient transfection experiments, HEK293 cells were transfected in 6 well dishes with 5 μg total DNA and 20 μl Superfect (Qiagen, 1:4 DNA:Superfect ratio).
Cloning and DNA manipulation
All PCRs were performed with Advantage DNA polymerase (Clontech). The E. coli gene for FlavoHb (hmpA) was amplified from genomic DNA (Strain BW25113) via PCR with the following primers: 5′-AATAGAATTCACCATGGACTACAAAGACGATGACGACAAGCTTGACGCTCAAACCA TCGCTAC-3′ and 5′-AAATGGATCCTTACAGCACCTTATGCGG-3′. The 1.2 kb PCR product was digested with EcoRI and BamHI, and ligated into the lentiviral expression plasmid pCDH-CMV-MCS-EF1-copGFP (System Biosciences) digested at the same sites. All products were verified by DNA sequencing (Duke DNA Sequencing Facility). Human iNOS was cloned into pCDH-CMV-MCS-EF1-copGFP at the XbaI and NotI sites using the following PCR primers: 5′-CTTATATCTAGAACCATGGCCTGTCCTTGG-3′ and 5′-ATTATAGCGGCCGCTTAGAGCGCTGACA-3’.
Lentiviral particles were generated by co-transfecting subconfluent (60–70%) 293FT cells with empty pCDH vector or pCDH-CMV-FlavoHb-EF1-copGFP plasmid DNA and the packaging and enveloping constructs, pCI-VSVG and ps-PAX2, respectively (Addgene). For a 10 cm dish, 6 μg plasmid DNA was co-transfected with 2 μg pCI-VSVG and 3 μg ps-PAX2 using 33 μL Lipofectamine 2000. Media were changed 8–12 hours after transfection to fresh DMEM with 10% FBS, and the virus-laden supernatant was collected at 24 and 48 hours. The supernatant was filtered and employed for experiments or frozen at −80 °C in aliquots.
Generation and isolation of cells stably infected with empty vs. FlavoHb lentivirus
Stable FlavoHb-expressing or vector control cells were generated by treating HEK293 or RAW264.7 macrophages twice with virus prepared as described above. Twenty-four hours after the second infection, HEK293 or RAW264.7 cells were dissociated using trypsin or cell scraping, respectively, and viable GFP-positive cells were single-cell sorted into 96-well cell culture plates containing DMEM using a FACS AriaII Cell Sorter (BD Biosciences). Single cell sorting was verified 24 hours after sorting and media changed on amplifying clones every 3 days until cell growth warranted splitting cells to a larger culture vessel. Amplified stable GFP-expressing cultures were evaluated for FlavoHb-expression by anti-Flagimmunoblotting.
Measurement of FlavoHb activity in cellular extracts via NO-dependent NADPH consumption
A 15 cm dish of HEK293 cells stably infected with either control lentivirus or FlavoHb was lysed via repeated passage through a 28 g needle in 50 mM PO4, 0.5 mM EDTA, 2 μM FAD, pH 7.4 containing 1 mM DTT and protease inhibitor cocktail (Roche). A cytosolic-enriched sample was obtained via clarification at 20,000 g × 30 min. Protein was measured via BCA assay. For measurement of NADPH consumption, 94 μl of lysate was mixed with either 2 μl of buffer or 10 mM NADPH, and absorbance at 340 nm was measured on a Beckman DU650 UV-Vis Spectrophotometer at 37 °C. Spermine-NONOate (final 4 mM, stock solution in 10 mM NaOH) was added to initiate the reaction, and absorbance at 340 nm was followed over time.
Subcellular fractionation for determination of FlavoHb localization in cells
Basic fractionation was achieved via differential centrifugation as briefly described. HEK293 cells were lysed via repeated passage through a 28 g needle in 25 mM Tris, 25 mM NaCl, 0.1 mM EDTA, pH 7.4 containing PI cocktail. Lysis efficiency was verified > 99% by trypan blue staining. The lysates were centrifuged at 500 g × 10 min to obtain a nuclei-enriched pellet. The supernatant was removed and centrifuged at 20,000 g × 30 min to obtain a membrane/organelle-enriched pellet and cytosol-enriched supernatant. To facilitate solubilization, lysis buffer was added to each fraction along with 0.2% Triton X-100 (v/v). Protein was measured by BCA assay, and equal amounts of material (40 μg each) were employed for immunoblotting analysis.
Measurement of NO2− and NO3− in culture media
Following the indicated iNOS transfection(s) or LPS-stimulations, culture media was collected and clarified by centrifugation at 5,000 g × 5 min. The supernatant was saved, and NO2− was measured by the standard Griess reaction. In brief, 40 μl of media or NO2− standard in culture media was transferred to a 96 well dish and 100 μl of 1% sulfanilamide (w/v) in 1 M HCl was added. The mixture was incubated at room temperature for 5 min, and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride (NEDD) in 0.5 M HCl was added to each well. Absorbance at 540 nm was measured via spectrophotometry and all samples were normalized to standard NO2− solutions. For measurement of NO3−, 40 μl of media or NO3− standard in culture media was transferred to two wells of a 96 well dish (labeled “A” and “B”). To all wells was added 40 μl of PBS containing 4 μM FAD, then 10 μl of 4 mM NADPH. To “A” and “B” wells was added 10 μl PBS or NO3− reductase (2 mU/μl in PBS), respectively. Plates were incubated at 37 °C for 1 h, and 100 μl of 1% sulfanilamide (w/v) in 1 M HCl was added. The mixture was incubated at room temperature for 5 min, and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride (NEDD) in 0.5 M HCl was added to each well. Absorbance at 540 nm was measured via spectrophotometry. The value of “B” minus “A” (i.e., NO3−-derived signal) was calculated for all samples/standards, and normalized accordingly.
Immunofluorescence of FlavoHb in mammalian cells
HEK293 cells were plated onto poly-D-lysine-coated coverslips in 6 well dishes. Upon reaching 50–60% confluence, cells were transfected with 2 μg pCDH-Flag-FlavoHb for 18 h, washed with PBS × 2, and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were again washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min on ice. Cells were blocked for 1 hour in PBS containing 5% goat serum, and stained overnight at 4 °C with PBS containing 5% goat serum and anti-flag rabbit pAb (1:50). Similar results were obtained with anti-Flag M5 mAb (data not shown) at 1:400 dilution. Cells were washed again with PBS and secondary anti-rabbit antibody with an Alexafluor-568 conjugate (Invitrogen) was added at 1:1000 dilution for 40 min at room temperature. Cells were washed repeatedly with PBS, mounted for imaging using mounting media and coverslips affixed to glass slides with nail polish adhesive. Cells were imaged on a Zeiss Axio Imager widefield fluorescence microscope (Duke Microscopy Core Facility) at 63x magnification.
For confocal imaging, HEK293 cells were plated onto tissue-culture treated chamber slides (Lab-Tek). Twenty-four hours after plating, cells were washed with PBS × 2 and fixed in paraformaldehyde (4% in PBS) for 15 minutes at room temperature. Cells were washed three times with PBS, and were permeabilized with 0.1% Triton X-100 in PBS and blocked with normal goat serum (10% in PBS) for 1 hour at room temperature. Slides were stained overnight at 4 °C with anti-Flag rabbit pAb (1:100), washed three times with PBS and incubated with Alexafluor-568 conjugate (Invitrogen) at 1:250 dilution for 20 min at room temperature. Cells were washed three times with PBS, were stained with Hoechst 33258 at 5 μg/mL for 10 min at room temperature before washing three times with PBS. Cells were coverslipped using FluorSave mounting media (Calbiochem) and imaged on a Leica SP5 confocal microscope, using sequential scanning technique with a 63x oil immersion objective.
Assessment of nitrosative stress by 3H-thymidine uptake, cell counting and colony formation assays
For evaluation of 3H-thymidine uptake, HEK293 cells were stably infected with either empty lentivirus or FlavoHb and viable cells were plated in equal numbers in 12-well plates (30,000 cells per well). Cells were treated with: 1) 0, 200, 500 or 1000 μM DETA-NO for 24 hours, or 2) were transfected with pCDH-CMV-iNOS-EF1-copGFP or vector control (pCDH-CMV-MCS-EF1-copGFP) for 12 hours before the media was changed to fresh DMEM + 10% FBS for 48 hours. At this point, cells were incubated for 5 h with 4 μCi of 3H-thymidine (Perkin Elmer), washed once with PBS, fixed for 1 hour in 10% trichloroacetic acid, and incubated overnight in 0.01 M NaOH before assessing radioactivity incorporation of the lysed cellular contents by scintillation counting (Beckman 156000).
For cell counting, HEK293 cells were stably infected with either empty lentivirus or FlavoHb and viable cells were plated in equal numbers in 6-well plates (100,000 cells per well). Cells were treated with 0, 200, 500 or 1000 μM DETA-NO for 48 hours, at which point cells were dissociated by trypsin, resuspended in a defined volume, and were mixed 1:1 with trypan blue for two minutes before viable cells were counted on a hemacytometer.
For colony formation assays, cells stably expressing either vector control or Flag-FlavoHb were plated at a density of 500 cells/well of a 6 well plate. DETA-NONOate was added at either 0, 0.1 or 0.5 mM final concentration and colonies were grown for 7 days before visualization by staining with 1% methylene blue in methanol (wt/vol) for 2 minutes.