Ja 10 rotor

FCS was cleared of cholesterol and other lipids by organic extraction as described [15] (link). Briefly, 100 ml of FCS was mixed with 200 ml of a mixture n-butanol and diisopropylether at a 40∶60 (v/v) ratio. After incubation at room temperature (22°C) for 1 hour in dark, the mixture was centrifuged at 22°C for 15 minutes at 6000 rpm in a JA-10 rotor, Beckman Coulter. The bottom phase containing delipidated serum was recovered and lyophilized. The resulting dry pellet was resuspended in 100 ml deionized H₂O and filter-sterilized through 0.22 µm filter. Concentration of cholesterol in serum and cell samples was determined by the Amplex Red Cholesterol Assay kit (Life Technologies) according to the manufacturer's instruction. Using this kit, no remaining cholesterol was detectable in delipidated serum. This indicates that cholesterol concentration was reduced from ∼80 µg/ml to <15 ng/ml. For determination of cellular cholesterol, frozen cell pellet containing 0.35×10⁶ cells was lysed in 30 µl of ice cold lysis buffer (10 mM EDTA, 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.2% SDS, 0.5% Nonidet P-40, 0.5% sodium deoxycholate) and 2 µl aliquots were analyzed for cholesterol content using the same kit as above. Protein content in the lysates was determined by BCA protein assay kit (Pierce Chemical Co.) and the amounts of cholesterol were normalized to protein contents.

We transformed the E. coli expression strain, BL21(DE3), with pEXS-CG-Annexin A2 (GST-tagged construct), and cultured the cells in Luria-Bertani (LB) liquid medium with 50 μg/mL ampicillin at 37 °C until the density reached an OD₆₀₀ of 1.5. Cells were cooled to 16 °C and cultured for another 12 h at 16 °C with 1.0 mM IPTG for protein expression. Then, cells were harvested by centrifuging at 6,000 rpm (JA10 rotor, Beckman) for 12 min. The cell pellet was resuspended in pre-cooled lysis buffer (PBS containing 1 mg/mL lysozyme, 1 mM DTT, and 1 mM PMSF), placed on ice, and ultra-sonicated for cell lysis. The lysed cells were separated with centrifugation at 12,000 rpm (JA25.50 rotor, Beckman) for 40 min, and the supernatant was loaded onto a GST-affinity chromatography column. The column was washed with pre-cooled PBS containing 1 M NaCl, 1 mM DTT, and 1 mM PMSF. Then, we added 10 μg of Human HRV 3 C Protease (TAKARA) to the column, and incubated the column at 4 °C overnight to allow cleavage of the GST-tag at the C-terminus of the protein. The eluted fraction was then loaded onto a Superdex75 16/600 column (GE Healthcare) and eluted at a flow rate of 1.0 mL/min. Fractions were pooled and concentrated to 10 mg/mL with a 10-kD cut-off centrifuge filter (Millipore).
The Annexin A2-K302A mutant was purified with the same procedure.

The E. coli expression strain, BL21 (DE3), was transformed with pEXS-CG-Annexin A2, and cultured in Luria-Bertani (LB) liquid medium with 50 μg/mL ampicillin at 37°C until the density reached an OD₆₀₀ of 1.5. Cells were cooled to 16°C and cultured for another 12 h at 16°C with 1.0 mmol/L IPTG for protein expression. Then, cells were harvested by centrifuging at 6,000 rpm (JA10 rotor, Beckman) for 12 min. The cell pellet was resuspended in pre-cooled lysis buffer (PBS containing 1 mg/mL lysozyme, 1 mmol/L DTT and 1 mmol/L PMSF), placed on ice, and ultra-sonicated for cell lysis. The lysed cells were separated with centrifugation at 12,000 rpm (JA25.50 rotor, Beckman) for 40 min, and the supernatant was loaded onto a GST-affinity chromatography column. The column was washed with pre-cooled PBS containing 1 mol/L NaCl, 1 mmol/L DTT and 1 mmol/L PMSF. Then, we added 10 μg of human HRV 3C protease (TAKARA) to the column, and incubated the column at 4°C overnight to allow cleavage of the GST-tag at the C-terminus of the protein. The eluted fraction was then loaded onto a Superdex75 16/600 column (GE Healthcare) and eluted at a flow rate of 1.0 mL/min. Fractions were pooled and concentrated to 10 mg/mL with a 10-kDa cut-off centrifuge filter (Millipore).
The Annexin A2-K302A mutant was purified with the same procedure.

Electrophoretic examination of genomic DNA extracted from H. ericina and P. pouchetii cultures was performed to investigate whether apoptotic DNA laddering (Nagata, 2000 (link)) occurs during viral infection of these phytoplankton. Culture samples (200–250 mL) were taken at various time points after infection (0, 1, 2 and 3 days post-virus addition). Cells were harvested by centrifugation at 1600–6400 × g at 4°C in a Beckman JA-10 rotor. Genomic DNA from cell pellets was immediately extracted using an Apoptotic DNA Ladder kit (Roche Applied Science, Indianapolis, IN, USA) according to kit instructions. After elution in 70°C elution buffer, RNA was removed from samples by the addition of 20 U RNAse A (Promega, Madison, WI, USA) and incubation at 37°C for 30 min. DNA was then purified using the Clean and Concentrator kit (Zymo Research, Irvine, CA, USA) and eluted in a final volume of 20 µL of 70°C distilled water. Four microliters of 6× DNA loading buffer (Promega) was added to each sample, and samples were stored at 4°C in the dark until analysis by agarose gel electrophoresis at 4°C in 1% (w/v) agarose at 100 V for 2 h in 40 mM Tris–Cl, 20 mM acetic acid, 1 mM EDTA, pH 8.0. Gels were stained in 1X SYBR Gold (Invitrogen, Carlsbad, CA) at room temperature in the dark for 1–2 h prior to visualization using a GelDocXR (Bio-Rad, Hercules, CA, USA).

The lrhA coding sequence was amplified using primers with BamHI and HindIII sites (Table S1), cloned into pGEM-T (Promega, Madison, WI, USA), and sequenced. After double digestion with BamHI and HindIII, the construct was ligated into pET28a (Novagen, Madison, WI, USA) and transformed into E. coli (BL21-DE3) (Studier & Moffatt, 1986 (link)) to express LrhA with a His₆ tag at the N-terminus (37 kDa). Induction of protein expression with 0.1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) was performed at an OD₆₀₀ of 0.5–0.8, 19 °C, overnight, shaking at 250 rpm. Cells were pelleted by centrifugation at 5,000 rpm in a JA-10 rotor (Beckman Coulter, Brea, CA, USA) for 20 min at 4 °C, snap-frozen with liquid nitrogen and stored at −75 °C. The cell pellet was then resuspended in Ni-NTA wash buffer (50 mM Tris–HCl, 300 mM NaCl, 50 mM imidazole) and sonicated to release proteins. Ultracentrifugation at 40,000 rpm in a Beckman Ti70 rotor for 1 h at 4 °C was used to subsequently remove the cell debris. The protein was purified using a Ni-NTA column (HisTrap HP, GE Healthcare) with Ni-NTA elution buffer (50 mM Tris–HCl, 300 mM NaCl, 500 mM imidazole). The protein purity was observed through standard SDS-PAGE electrophoresis.