Plasmids Western blot analysis and immunostaining Flow Cytometry Cell surface biotinylation RNA isolation and cdna synthesis

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Augustus Young
6 years ago
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1 Plasmids psuper-retro-s100a10 shrna1 was constructed by cloning the dsdna oligo 5 -GAT CCC CGT GGG CTT CCA GAG CTT CTT TCA AGA GAA GAA GCT CTG GAA GCC CAC TTT TTA-3 and 5 -AGC TTA AAA AGT GGG CTT CCA GAG CTT CTT CTC TTG AAA GAA GCT CTG GAA GCC CAC GGG-3 into psuper.retro.puro (OligoEngine), psuper-retro- S100A10 shrna5 was constructed by cloning the dsdna oligo 5 -GAT CCC CAG AGT ACT CAT GGA AAA GGT TCA AGA GAC CTT TTC CAT GAG TAC TCT TTT TTA-3 and 5 - AGC TTA AAA AAG AGT ACT CAT GGA AAA GGT CTC TTG AAC CTT TTC CAT GAG TAC TCT GGG-3 into psuper.retro.puro (OligoEngine) and the psuper-retro-s100a10 scramble was constructed by cloning the dsdna oligo 5 -GAT CCC CGT GGG AGT TCA GAG CTT CTT TCA AGA GAA GAA GCT CTG AAC TCC CAC TTT TTA-3 and 5 -AGC TTA AAA AGT GGG AGT TCA GAG CTT CTT CTC TTG AAA GAA GCT CTG AAC TCC CAC GGG-3 into psuper.retro.puro (OligoEngine). Western blot analysis and immunostaining Cells were lysed with cell lysis buffer (1% NP-40, 150 mm NaCl, 20 mm Tris-HCl, 1 mm EDTA, 1 mm EGTA, and proteinase inhibitor cocktail (1:500; Sigma-Aldrich)). 20 μg total protein was loaded into each well and was resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes. Membranes were incubated with antibodies for S100A10 (BD Biosciences), annexin A2 (BD Biosciences), α- enolase (Abcam), histone 2B (Abcam), RARα (Santa Cruz) and actin (loading control; Sigma) and the secondary IRdye-800 antibody (LI-COR Biosciences). Antibody complexes were viewed on the Odyssey IR imaging system (LI-COR Biosciences). Protein expression was quantified using the Odyssey quantification software. Proteolyzed annexin A2 was prepared according to Kwon et al. 32 Flow Cytometry To assess surface expression of annexin A2 and S100A10, cells were Fc blocked with mouse IgG and then stained with the appropriate antibodies in PBS containing 0.1% sodium azide. Cells were further stained with fluorescein isothiocyanate (FITC) goat anti-mouse antibody (Sigma- Aldrich). Cell fluorescence was measured by fluorescence-activated cell sorter (FACS). Cell surface biotinylation Cells were washed with incubation buffer (IB; 20 mm HEPES, 3 mm CaCl 2, 1 mm MgCl 2, and 150 mm NaCl) and incubated with 1 mm Sulfo-NHS-SS-biotin (Pierce, Rockford, IL) in IB for 30 min at room temperature. Cells were lysed in lysis buffer (see western blot analysis above) on ice for 10 min. 100 µg total protein was incubated with 30 μl of Dynabeads M-280 streptavidin (Invitrogen, Carlsbad, CA) for 2 hours at 4 o C with rotation and then washed 5 with lysis buffer. Beads were resuspended in 2 SDS-PAGE loading buffer, electrophoresed, and immunostained for S100A10, and annexin A2. RNA isolation and cdna synthesis Total RNA was isolated with the Aurum Total RNA Mini Kit (Bio-Rad, CA, USA) as per manufacturer s instructions. RNA was pre-incubated with random hexamer primers at 70 C for 10 minutes and the reverse transcription reaction was performed at 42 C for 60 min. Reverse

2 transcription reactions contained 1 μg of total RNA, 4 PCR buffer (Invitrogen), 1 mm deoxynucleotide triphospates datp, dgtp, dctp and dttp (Invitrogen), 40 units RnaseOUT (Invitrogen), 2 μl DTT (Invitrogen), 100 pmol of random hexamer primers (Invitrogen) and 200 units of Super script II reverse transcriptase (Invitrogen). Quantitative real-time PCR For PCR reactions, specific primers and fluorogenic probes for S100A10 and annexin A2 were designed using IDT Primer Quest software (PE Applied Biosystems) and synthesized by IDT. Q- PCR was performed by the use of the Rotor-Gene 6000 (Corbett Life Science). The reaction was performed in a final volume of 20 µl by the use of 10 µl SsoFast EvaGreen Supermix (Bio-Rad, CA, USA) 1 µl of each 3 and 5 primer (IDT), and 0.5 ng/µl cdna. Amplification was performed after initial incubation at 95 C for 10 minutes in a 3-step cycle procedure (denaturation 95 C, 10 second, annealing temperature 60 C, 15 seconds, and extension 72 C, 20 seconds, ramp rate 2 C/s) for 45 cycles. Primer Sequences are as follows: S100A10 forward primer 5 -TAG AGA TGG CAA AGT GGG CTT CCA-3 S100A10 reverse primer 5 -TCC TTA AGC GAC CCT TTG GGA CAA-3 Annexin A2 forward primer 5 -ACT TTG TGG CCC TGC TTT CAA CTG-3 Annexin A2 reverse primer 5 -ACA GCT CAG TCC CAG AGC TTT CTT-3 The expression of the S100A10 and annexin A2 was normalized against the expression of the control gene GAPDH to adjust for variations in mrna quality and efficiencies of cdna synthesis. FITC-plasminogen preparation Glu-plasminogen (2 5 mg/ml) was dialyzed against 0.1 M carbonate buffer (ph 9) and a 50 molar excess of FITC was added after being dissolved in DMSO. Plasminogen and FITC were mixed for 16 hr in the dark and treated with 0.01% hydroxylamine to remove all labile FITCplasminogen bonds. Unincorporated FITC was removed by gel-filtration through a NAP-10 column using HBSS (20 mm HEPES, 1 mm CaCl 2, and 1 mm MgCl 2; ph 7.4). Typically, 4 FITC molecules were bound to each plasminogen molecule.

3 Figure S1. Plot of the relative quantities of S100A10 and annexin II mrna in NB4 and PR9 cells using Real-time PCR Real-time PCR was performed on the cdna product following reverse transcription of RNA extracts. Results are expressed as relative quantities of S100A10 and annexin II mrna in comparison to non-treated (NT) cells. Values are expressed as means ± standard deviation. NB4 cells were treated with ATRA and RNA extracts were prepared at the indicated time points. Relative S100A10 (A) and annexin A2 (B) mrna levels were examined. PR9 cells were induced with 100 µm ZnSO4 for the indicated times. RNA extracts were prepared and relative S100A10 and annexin A2 mrna levels were examined.

4 Figure S2. Cell surface annexin A2 on S100A10-depleted NB4 and PR9 cells Cell surface proteins of S100A10-depleted NB4 and PR9 cells were incubated with Sulfo-NHS-SS-biotin, lysed and the biotinylated (cell surface) proteins were collected with streptavidin beads and subjected to SDS PAGE and Western blotting for annexin A2. Total and cell surface annexin A2 obtained from S100A10-depleted NB4 (A) and PR9 (B) cells were compared with proteolyzed recombinant annexin A2. PR9 cells were examined in the absence or presence of ZnSO4.

5 Figure S3. Plasmin generation by S100A10-depleted and annexin A2-depleted PR9 cells PR9 cells were transduced with a retroviral shrna system using shrna specific for two sequences of S100A10 (shrna 1 and shrna 5), two sequences of Annexin A2 (shrna 3 and shrna 4) and a shrna scramble sequence (shrna Scr). Cell lysates were prepared and total levels of S100A10 and annexin A2 was examined by Western blotting using actin as a loading control (A). PR9 cells were incubated with upa (50 nm) and plasminogen (0.5 μm) and the rate of plasmin generation was measured at 405 nm. Statistical analysis was performed by student s t-test (***p<0.001) and data is expressed as Δ405 nm/sec plus or minus SD of 3 independent experiments (B).

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative