PMC

A & B) Pie charts representing annotated biological process of proteins identified as regulators of SOD1. Contribution of each biological process as a percentage of total are shown. Contributions of processes that are less than 2% of the total are not shown in the figure. C) Effect of siRNA knockdowns of SOD1 isoforms in the screen. The MAD score values of the three SOD1 isoforms obtained in the screen are shown. The error bars represent SD.

A) Luminescence intensity measurements of the genome wide RNA screen of 21125 wells in triplicates were processed by MAD score analysis method and the results are plotted against well numbers. Horizontal dotted lines indicate the hit selection criteria of +3 MAD or −2MAD. The two numbers listed in the plot represent the total number of hits that meet the two selection criteria. B) Luminescence intensity measurements in triplicates were processed by Z score analysis method and the results are plotted against well numbers. Horizontal dotted lines indicate the hit selection criteria of +3 SD or −2SD. The numbers listed in the plot represent the total number of hits that meet the hit selection criteria.

A) TDP-43 knockdown or over expression was carried out in HeLa TetOn cells expressing the A4V reporter plasmids. Supernatant fractions were assayed for β-gal activity. Soluble SOD1 reporter expression levels (β-gal activity) relative to controls (dotted line) are shown. Sample sizes ranged from 8 to 23. The error bars represent 95% confidence interval. B) Quantitative PCR analysis of SOD1 reporter mRNA levels was carried out on total mRNA isolated from cells transfected with control or TDP-43 siRNA and A4VSOD1 reporter plasmids. The SOD1 mRNA levels of TDP siRNA treated samples are shown relative to control (dotted line). The error bars represent 95% confidence interval.

The data from the RNAi screen was processed to calculate MAD scores and was used for Ingenuity pathway analysis (IPA). The network annotated “Skeletal and Muscular System Development and Function, Tissue Morphology, Inflammatory Response" ranked at the top with the highest score and is shown in the figure. The hits that increased SOD1 levels above 3SD are shown in red and the ones that decreased the signal below −2SD are shown in green. The proteins represented by color-less nodes were below the selection criteria. Direct interactions are shown in bold lines and indirect interactions are shown in broken lines. (See for proteins in this network)

A) Effect of knock down of a proteasomal subunit on SOD1 levels. HeLa cells were transfected with siRNA targeting the S4 subunit (PSMC1) of the 26 S proteasome, followed by expression of the mutant SOD1 reporter plasmids. Supernatant fractions were tested for β-gal activity by measuring the fluorescence signal. The plot shows SOD1 levels as a measure of β-gal activity. B) HeLa cells were co-transfected with S4 siRNA or carrier and varying amounts of plasmids coding for A4VSOD1-HA-α fusion with a fixed amount of ω fragment. Cells were analyzed for fluorescence 96 hrs post transfection. The SOD1 levels measured as a function of β-gal activity are plotted against each transfection condition. Z′ factor was used to assess the assays suitability for HTS.