Eucaryotic cell culture and transfections
SH-SY5Y neuroblastoma cells [14] were obtained from ATCC, Manassas, USA and grown at 37°C and 5% CO2 in Dulbecco's modified Eagle's Medium containing 1% penicillin/streptomycin and 15% fetal calf serum (growth medium; GIBCO, Eggenstein, Germany) in a humidified incubator. To keep the cells in logarithmic growth, confluent cells were washed with phosphate buffered saline (PBS), detached by 0.05% Trypsin and 0.5 mM EDTA in PBS, followed by washing and diluting them in fresh growth medium. 106 cells were transfected with 2 μg Plasmid by electroporation in 50 μl of Cell Line Nucleofector V solution (Amaxa, Cologne, Germany). After transfection, cells were washed in growth medium, transferred to 6-well plates and judged microscopically after 24 h of incubation at 37°C and 5% CO2 using an Axiovert100 microscope (Zeiss, Oberkochen, Germany). Red fluorescent cells were analysed at 584 nm and green fluorescent cells at 482 nm excitation. Three different fields of view were evaluated for each experiment and all experiments were performed independently in triplicate.
Silencing HCR-NTPase in SH-SY5Y by RNA interference
The expression of HCR-NTPase in SH-SY5Y cells was blocked via RNA interference [15] using pSilencer1.0-U6 (Ambion, Darmstadt, Germany). shRNA coding inserts flanked by ApaI and EcoRI restriction sites were constructed by phosphorylating 25 pmol of oligonucleotides (Operon, Cologne, Germany) using T4 poynucleotide kinase, followed by assembling complementary pairs by incubation for 5 min at 100°C and 60 min at 37°C. pSilencer1.0-U6 sequentially was cut by ApaI (Fermentas, St. Leon-Rot, Germany) and EcoRI (Fermentas, St. Leon-Rot, Germany), the inserts were inserted and the plasmids were amplified in E. coli SURE (Stratagene, San Diego, USA). Expression of HCR-NTPase was blocked by transfecting SH-SY5Y cells with purified plasmids. The following pairs of oligonucleotides were used:
siRNA1:
5'-ATC CAT AAA GCC AGT GAT TCT CAA GAG AAA TCA CTG GCT TTA TGG ATC ATT TTT T-3' and
5'-AAT TAA AAA ATG ATC CAT AAA GCC AGT GAT TTC TCT TGA GAA TCA CTG GCT TTA TGG ATG GCC-3'
siRNA2:
5'-AGA GCC TCC ACC TGG AAT TCT CAA GAG AAA TTC CAG GTG GAG GCT CTA ATT TTT T-3' and
5'-AAT TAA AAA ATT AGA GCC TCC ACC TGG AAT TTC TCT TGA GAA TTC CAG GTG GAG GCT CTG GCC-3'
siRNA3:
5'-GAA TGC CGA CTG CAG CAT TCT CAA GAG AAA TGC TGC AGT CGG CAT TCC TTT TTT T-3' and
5'-AAT TAA AAA AAG GAA TGC CGA CTG CAG CAT TTC TCT TGA GAA TGC TGC AGT CGG CAT TCG GCC-3'
siRNA4:
5'-TTC CTA AAG GAA AGC CAT TCT CAA GAG AAA TGG CTT TCC TTT AGG AAC TTT TTT T-3' and
5'-AAT TAA AAA AAG TTC CTA AAG GAA AGC CAT TTC TCT TGA GAA TGG CTT TCC TTT AGG AAG GCC-3'
Overexpressing HCR-NTPase in SH-SY5Y cells
HCR-NTPase cDNA was obtained from RZPD, Berlin, Germany. The gene was amplified and cloned into pET101/D-TOPO yielding pET101/D-TOPO/HCR-NTPase to express the protein in E. coli. This construct also served as the basis for all further cloning steps. HCR-NTPase was overexpressed using the plasmid pRc/CMV (Invitrogen, Karlsruhe, Germany). The gene was amplified from pET101/D-TOPO/HCR-NTPase using the primers
5'-AAA AGC TTA TGG CCC GGC ACG TGT TCC-3' and
5'-AAA ATC TAG ATC ACT TCC TGC TGC TCT G-3'.
The PCR-fragment was digested sequentially by XbaI (Fermentas, St. Leon-Rot, Germany) and HindIII (Fermentas, St. Leon-Rot, Germany), inserted into pRc/CMV and amplified in E. coli TOP10 (Invitrogen, Karlsruhe, Germany).
HCR-NTPase as a fusion with red fluorescent protein [16] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany). The gene was amplified from pET101/D-TOPO/HCR-NTPase using the primers
5'-AAA AGA TCT GGA GGA GGA GGA ATG GCC CGG CAC GTG TTC-3' and
5'-AAG GTA CCT CAC TTC CTG CTG CTC TG-3'.
Before digesting with BglII (Fermentas, St. Leon-Rot, Germany) and KpnI (Fermentas, St. Leon-Rot, Germany), the PCR-fragment was inserted into pCR2.1-TOPO (Invitrogen, Karlsruhe, Germany). The resulting fragment was inserted into pmaxFP-Red-C and amplified in E. coli TOP10. As a control for the efficiency of transfection, pmaxGFP (QIAGEN) was used. HCR-NTPase was overexpressed by transfecting SH-SY5Y cells with purified plasmids.
Construction of a E114A-HCR-NTPase mutant in pRc/CMV
An enzymatically inactive HCR-NTPase mutant was constructed by mutating a conserved catalytic glutamate [17]. For that purpose, E114 of HCR-NTPase in pRc/CMV was replaced by an alanin using the Phusion™ Site-Directed Mutagenesis Kit (Finnzymes, Espoo, Finland) according to the instructions of the manufacturer. The utilised phosphorylated primers were
5'-P-CGT CAT CGA TGC GAT TGG GAA GA-3' and
5'-P-CAC ACT CTT TGC CCT GGG CCA-3'.
Inhibition of apoptosis
Apoptosis was inhibited by adding the caspase inhibitor Z-VAD-FMK (Promega, Mannheim) to the culture medium directly after transfection at a final concentration of 20 μM.
Bioinformatics
ClustalW-alignments were performed as previously described [18, 19] and for the graphical representation, Genedoc was used [20].
All investigations have been performed in accordance to German regulatory affairs.