PMC

RT-PCR analysis of SOD transcripts in HeLa and MCF-7 cells treated with siRNA against CLK-1. (A) Results of amplification of SOD1 cDNA using primers spanning exon 1 through 3, exon 3 through 5 and and exon 1 through 5 (ntc: no template control).

Knockdown of CLK-1 increases SOD activity in HeLa and MCF-7 cells. (A) Total SOD activity in HeLa cells treated with siRNA against CLK-1, CLK-2, CLK-3 and CLK-4. (B) SOD1 activity in HeLa and MCF-7 cells treated with CLK-1 siRNA. (C) SOD2 activity in HeLa and MCF-7 cells treated with CLK-1 siRNA.. White bars indicate non-targeting siSCR controls; black bars indicate siCLK-1. In each assay RT-PCR confirmed that the target transcript level was reduced at least 80%. Error bars indicate standard error. *SOD activities significantly different from control values (p < 0.05).

Knockdown of CLK-1 increases SOD transcript and protein. (A) Western blots of lysates from HeLa cells and MCF-7 cells treated with CLK-1 siRNA. (B) SOD1 and SOD2 transcript levels in MCF-7 cells treated with CLK-1 siRNA were measured using Taqman real-time PCR. (C) SOD1 and SOD2 transcript levels in HeLa cells treated with CLK-1 siRNA were measured using Taqman real-time PCR. (D) Western blots of lysates from HeLa cells and MCF-7 cells grown for 48 hr in 10 μM TG003. In each assay RT-PCR confirmed that the target transcript level was reduced at least 80%. Error bars indicate standard error. *SOD- transcripts significantly different from control values (p < 0.05). Staining for β-actin was done to control for sample loading.

Mutations in Doa cause resistance to paraquat. Ore R, Doa^γ3B/+ and Doa^HD/+ animals 1–3 days post eclosion were starved for 6 hr, and then fed continuously with 10 mM paraquat dissolved in 5% sucrose. The number of dead animals was counted twice each day for 5 days and the data was expressed as percent survival. The estimated times of 50% mortality were 74.3 h for Ore R, 109.2 h for Doa^γ3B/+ and 116.2 h for Doa^HD/+. Less than 1% of the sibling control animals for each genotype that were fed 5% sucrose without paraquat died during the 5-day counting period. n ≥ 100 for each genotype.

SOD activity is elevated in adult flies carrying mutations in Doa. (A) Total SOD activity was measured in homogenates of age-matched Doa^γ3B/+ and Doa^HD/+ males. Ore R males were assayed as wild type controls. SOD2 activity was measured in parallel aliquots containing 5 mM NaCN. SOD1 activity was then estimated by subtracting the SOD2 activity from the total activity. (B) Representative Western blot of homogenates of adult males 1–3 days post eclosion. Lane 1, wild type (Ore R); lane 2, Doa^γ3B/+; lane 3, Doa^HD/+. Staining for actin is included on all Western blots as a control for loading. (C) SOD activity in trans-heterozygous Doa^Dem/Doa^HD flies along with heterozygous Doa^Dem/TM6B and Doa^HD/TM6B siblings. Because the Doa^Dem/TM6B and Doa^HDTM6B animals were in yw and w^a backgrounds, respectively, stocks of yw and TM6B/TM3 animals were used as controls. Error bars indicate standard error. * SOD activities significantly different from control values (p < 0.05).