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. 2023 Jul 22;26(8):107375. doi: 10.1016/j.isci.2023.107375

Figure 4.

Figure 4

Overexpressing SIRT3 plus PARP inhibition with olaparib further reduces ROS in vitro and rescues treadmill running without affecting AR100Q protein levels

(A) ROS in AR112Q SIRT3-MYC-overexpressing PC12 cells (n = 12) treated with either DMSO (vehicle control), or 1 μM olaparib for 48 h with (right) or without (left) ROS induction with 250 μM TBHP (representative image from 3 experiments).

(B) Muscle mass and percent of total mass of quadriceps (left) and gastrocnemius (right) from WT, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 8 weeks fed no-drug compounded or olaparib-compounded feed.

(C) Behavioral cohort composed of 8 X WT fed no-drug compounded food, 11 X AR100Q fed no-drug compounded food, and 11 X AR100Q-SIRT3 male mice fed olaparib-compounded food. Left, accelerating rotarod analysis starting at age 6 weeks; ∗ = difference between WT vs. AR100Q and AR100Q-SIRT3. Right, accelerating treadmill analysis at 11 weeks of age.

(D) Left, immunoblot of total PAR following a PAR affinity resin assay from 8-week-old mouse quadriceps. WT lanes are oversaturated. Right-top, analysis of total protein lysates utilized in the PAR assay showing AR100Q monomer, and SDS-insoluble HMW aggregated AR100Q and tubulin loading control. Right-bottom, densitometry analysis of AR monomer and SDS-insoluble HMW aggregated AR normalized to tubulin loading control.

(A–D) All statistical analysis show mean ± SD, and used a two-way ANOVA and Tukey’s post-hoc (∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001). TBHP, tert-butyl hydroperoxide; WT, wild type; AR100Q, polyQ-expanded AR; AR100Q-SIRT3, polyQ-expanded AR with SIRT3-flag overexpression; HMW, high molecular weight; olaparib, PARP inhibitor (1150 g of olaparib in 1 kg of feed); PAR, poly-ADP-ribose.