D. discoideumculturing conditions
D. discoideum Ax-2 strain which is an axenic derivative of Raper's wild type NC-4 (a mutant in at least two genes i.e. axe A and axe B) was used. D. discoideum was grown under different culture conditions. The growing cells (unicellular) were maintained in a liquid suspension (HL5 medium). D. discoideum cells were grown in HL5 medium, pH 6.5 with 150 rpm shaking at 22°C . Log phase cells at a density of ~2.5 × 106 cells/ml were used for experiments.
D. discoideum was maintained on a solid substratum containing Phosphate Buffered Agar (PBA). D. discoideum was also cultured on bacterial lawn of Klebsiella which is its natural food. For this a loop full of overnight grown culture of Klebsiella was taken and D. discoideum spores (4-5) were mixed with it. This 'mixture' of two cell types is then pour plated on PBA plates (90 mm). D. discoideum cells fed on Klebsiella and when no more Klebsiella was left the cells undergo developmental changes and form fruiting bodies.
Monitoring growth of D. discoideumcells
1.5 × 106 cells were harvested, washed with 1X Sorenson's buffer (SB) and finally resuspended in 4 ml of sterile HL5 containing flask and growth was monitored for 6 days at an interval of 12 hours under shaking conditions at 22°C.
Induction of oxidative stress
Oxidative stress was induced in D. discoideum cells by in situ generation of H2O2 upon addition of hydroxylamine (HA) (Sigma), . Log phase cells at a density of ~2.5 × 106 cells/ml were exposed to different doses of HA (0, 1, 2.5, 4 mM) in HL5 medium at 22°C in a sterile flask.
Effect of benzamide on the development of D. discoideumcells
1.5 × 106 cells were treated with the benzamide (0, 1, 2, 3, 4, 6, 10 mM) for 12 hours. Following this, the cells (pre-exposed to benzamide) were then washed with 1X SB and the pellet was resuspended in 100 μl of 1X SB and spread on PBA plate . Different developmental stages were monitored at an interval of 6 hours.
Expression analysis of aca, cAR1, yakA, parp, countin50, gp80 and hspD by RT-PCR
D. discoideum cells were exposed to oxidative stress as mentioned earlier. After one hour pretreatment cells were pelleted and washed with 1X SB and finally resuspended in 1X SB. Total RNA was isolated from the cells at two time points (6 and 10 hours) using TRIZOL reagent (Invitrogen, USA). The expression kinetics of acaA, cAR1, yakA, parp, countin50, gp80 and hspD was examined by RT-PCR and rnlA was used as an internal control. The reactions were performed according to the manufacturer's instructions (Fermentas, Ontario, Canada). DNA fragments were amplified for 24 cycles after reverse transcription and signal intensities were analyzed on 2% agarose gel.
Strategy for targeted down-regulation of adprt1A encoding PARP
Antisense of 500 bp was designed for the catalytic domain of PARP. This region was PCR amplified using oligonucleotide primers (left primer: 5'AAAACGGGTTCCTCACTTTG3' and right primer: 5'CGGCGATTAGAATTCTTCGT 3'). The confirmed amplified product was cloned in Bluescript KS+ vector. Randomly selected white colonies were screened for the presence of recombinant plasmid which was confirmed by restriction digestion pattern. This recombinant plasmid was used as an intermediate plasmid for further cloning in the target vectors (constitutive and stage specific). For cloning in pTX vector, the intermediate recombinant plasmid was digested with KpnI and BamHI and ligated with KpnI and BamHI digested pTX. Presence of PARP antisense insert was confirmed by colony PCR and relevant restriction enzyme digestion patterns. The confirmed clone containing PARP antisense, pTX-PARP, was used for transformation of D. discoideum cells.
Similar strategy was also followed for cloning PARP antisense in a stage specific vector EcmB using SmaI and XhoI enzymes. Clones obtained were screened by relevant restriction enzyme digestion and confirmed by PCR and was named as EcmB-PARP. The confirmed clones i.e., pTX-PARP and EcmB-PARP were independently used to generate D. discoideum transformants with constitutive and inducible down-regulation of PARP respectively.
Measurement of PARP activity by indirect immunofluorescence 
PARP activity was assayed by using antibodies against the product of PARP i.e. PAR. For assaying PARP, indirect immunofluorescence was done using anti-PAR mouse mAb (10 H) (Calbiochem, Germany) at a concentration of 0.5 μg/ml and anti-mouse IgG (secondary antibody)-FITC conjugate (Sigma) at a dilution of 1:200. D. discoideum cells were pelleted and washed once with phosphate buffered saline (PBS) pH 7.4, fixed in 70% chilled methanol for 10 minutes at -20°C and then washed with blocking solution (1.5% BSA with 0.05% Tween 20 in PBS) followed by incubation for 1 hour in primary antibody. After incubation the cells were washed 2-3 times with blocking solution and further incubated for 1 hour with FITC labeled secondary antibody. Finally these cells were washed 2-3 times with PBS and the fluorescence was observed at 490 nm under 60X magnification.