Figure 9
Colchicine and doxorubicin effects on TDP-43 and TDP-25 aggregation (A) Representative immunofluorescence microscopy analysis (63x magnification and 2.5x zoom) performed in SH-SY5Y cells transiently transfected for 24 hrs with GFP-TDP-43 or GFP-TDP-25 plasmids and then treated for 24 hrs with 1 μM colchicine, doxorubicin or DMSO.
Left panels: GFP-TDP-43; right panels: GFP-TDP-25. DAPI: nuclei staining. RED: HSPB8. (B) Quantification of GFP-TDP-43 and GFP-TDP-25 positive cells with GFP-spots using Operetta Imaging System and Harmony software analysis (Perkin Elmer) on living SH-SY5Y cells transiently transfected and treated as described in A. Spots are defined with a range of size from 1 to 10 μm2 in SH-SY5Y treated as in A. SD refers to four independent replicates in HCS. (*p < 0.05 and **p < 0.01 vs untreated control). (C) Quantification of spots area in GFP-TDP-43 and GFP-TDP-25 positive SH-SY5Y cells after drugs treatment obtained by Harmony software analysis (Perkin Elmer).(n = 4). (*p < 0.05 and **p < 0.01 vs untreated control). (D,E) Representative FRA and FRA quantifications of the PBS extracts of SH-SY5Y cells transiently overexpressing the GFP-TDP-43 or GFP-TDP-25 plasmids treated or not with 500 nM of colchicine (left insets) or doxorubicin (right insets) the day after transfection, for the last 24 hrs prior to extraction. (D) The histograms represent densitometric analysis of three independent replicates for each sample (**p < 0.01 vs untreated GFP-TDP-43; °p < 0.05 vs untreated GFP-TDP-25). (E) The histogram represents the quantification of the GFP-TDPs insoluble species reduction percentage after treatment with colchicine or doxorubicin.
