Fig 1: Fodrin cleavage is reduced by TAT-MTScs-FXN treatment (A) Western blotting of a-Fodrin fragmentation. Crude extracts from Scr, Fxn1 and Fxn2 cells treated with increasing doses of 1, 3 and 7 µg/ml TAT-MTScs-FXN or vehicle solution 48 hrs after lentiviral transduction were checked for a-Fodrin fragmentation (ß-actin was used for normalization). TAT-MTScs-FXN caused reduction in a-fodrin cleavage as judged by the decrease of fodrin fragments (150 and 120 kD) in a dose-dependent manner. (B) Histogram showing the decrease in relative amounts of fragmented a-fodrin shown in A as function of the amount of TAT-MTScs-FXN added to cultures. (C) a-fodrin cleavage after 5 days analysed in Scr, Fxn1 and Fxn2 cells treated with 7 µg/ml TAT-MTScs-FXN or vehicle solution 12 or 48 hrs after lentiviral transduction. The histogram in (D) represents the values of fodrin fragments shown in (C) to illustrate the differences of adding the compound at 12 or 48 hrs. Values are referred to untreated control (Scr) culture. In (E), procaspase 9 levels are shown in Scr, Fxn1, Fxn2 treated with TAT-MTScs-FXN and untreated. Note that the decreased amounts in untreated Fxn1 and Fxn2 are re-established by TAT-MTScs-FXN addition. The histogram in (F) represents the values shown in (E). Asterisks (*) show significant values compared with control conditions (Scr); bars with # indicate significant differences with respect to the corresponding untreated control culture. Error bars represent mean ± S.E.M. In all figures, N is at least n = 4.
Fig 2: Cell survival is enhanced by TAT-MTScs-FXN treatment. (A) shows the percentage of surviving DRG neurons at day 5 of cultures treated with TAT-MTScs-FXN (7 µg/ml, 12 hrs post-lentivirus transduction) compared to untreated cells. Note the significant survival increase by TAT-MTScs-FXN supplementation in both Fxn1 and Fxn2 cultures. Error bars represent mean ± S.D., n = 4, and a range of 956–1184 neurons was scored for Scr conditions and 1397–1733 for each Fxn1 and Fxn2 conditions. (B) Representative images of neuronal survival with or without treatment (corresponding to 1/4th of full-size field). Images were taken at the same fields of cultures at day 0 and day 5. Arrows indicate the neuronal bodies present at day 0 and absent at day 5. In (C), the effect on survival of DRG-deficient neurons when treated with TAT-MTScs-FXN added at 48 hrs after lentivirus transduction is shown. Significance is marked as (*) when referred to no treatment in Scr cultures and (#) when comparing 1, 3 and 7 µg/ml treatments. Error bars represent mean ± S.D., n = 4, a range of 436–1233 neurons were scored for Scr condition and 867–1832 for each Fxn1 and Fxn2 conditions.
Fig 3: Frataxin detection in cardiac tissue of MCK mice model. Immunohistochemical staining of frataxin from WT, MCK vehicle-treated and MCK TAT-MTScs-FXN-treated mice are shown. Arrows indicate Frataxin accumulation near intercalated discs of cardiomyocytes for WT and MCK TAT-MTScs-FXN-treated mice, but no frataxin were detected in MCK vehicle-treated mice.
Fig 4: FRDAkd mice induced by doxycycline-compounded feed display FRDA-like phenotype (A) Survival rate of the FRDAkd mice after dox-feed induction. (TG + , n = 51; TG-, n = 27; TG ± , n = 13; WT + , n = 44; WT-, n = 28; WT ± , n = 14). (B) Animal weight throughout dox treatment and reversal in all groups (n = 21 TG + , n = 6 TG-, n = 13 TG ± , n = 17 WT + , n = 14 WT-, n = 14 WT ±). (C) Rotarod analysis throughout dox treatment and reversal in all groups TG ± , n = 8; TG + , n = 6; WT ± , n = 8; WT + , n = 8. Stats for panels C and D: Two-way repeated-measures ANOVA with Tukey’s post hoc (p values in Table 2). (TG + = transgenic with dox treatment for 18 weeks, TG- = transgenic no dox treatment, TG ± = transgenic with dox treatment for 9 weeks and reversal for subsequent 9 weeks, WT + = wild-type with dox treatment for 9 weeks, WT- = wild-type no dox treatment, WT ± = wild-type with dox treatment for 9 weeks and reversal for subsequent 9 weeks.) At week 0, all mice were between 2 and 5 months of age.
Fig 5: Compounds identified from the screen show efficacy in improving Fxn levels in doxorubicin-treated cardiomyocytes.A, H9C2 rat cardiomyocytes were cultured in the presence of doxorubicin and 10 to 100 µM DBM for 48 h and Fxn and GAPDH levels determined by Western blot. A representative blot is shown. Quantification of three biologic replicate experiments is shown. B, quantification of changes in response to other top compounds from the screen are shown (n = 3 replicate experiments). DBM, dibenzoylmethane; Fxn, frataxin.
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