<p>A) Manual quantitation of the electron micrographs using NIH Image J to identify and hand measure the length of each filament indicates the number of filaments reaches steady state at T = 120 minutes. B) This is reflected in the total summed filament length per image which also reaches equilibrium at T = 120 minutes. C) The average length of these filaments does appear to grow from the thresh hold limit of 80 nm at T = 5 minutes to nearly 200 nm at T = 120 minutes. Here the # indicates p<0.10 and the * indicates p<0.01 compared to T = 5 minutes values. The % indicates p<0.10 for the T = 120 and T = 180 minute lengths perhaps indicating a length redistribution. D) Similarly, there is a modest increase in Thioflavin S fluorescence with the addition of CKII that is 1.9 X higher at T = 180 minutes than the monomeric rTDP-43 assayed at T = 0 minutes, and again the * indicates p<0.01 significance. This was a larger increase than the 1.3 X fold Thioflavin S fluorescence increase detected due to aggregation alone presented earlier. E) Similarly there was only a modest 1.3 x fold increase in turbidity between the buffer controls (diamonds) and the CKII treated TDP-43 samples (circles) again indicating quantitation of the electron micrographs provides the most accurate representation of the soluble rTDP-43 polymerization process. F) Image Pro plus was able to identify a very similar but lower numbers of filaments compared to Image J manual counting as indicated by a slope of less than 1.0 in the best fit line where IPP = 0.898 (NIJ) + and R<sup>2</sup> = 0.578. G) Also the total filament length was slightly underestimated using Image Pro Plus where IPP = 0.812 (NIJ) + 265 and R<sup>2</sup> = 0.664. This provides a significant increase in counting efficiency even with the manual IPP masking.</p
