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

Figure 1.

Figure 1

SIRT3 and SOD2 proteins are reduced in AR100Q mice, and activating and increasing SIRT3 reduces ROS and cell death in vitro

(A) Western analysis of quadriceps from 11 week-old WT, AR24Q, and AR100Q male mice (n = 3). Top, total SOD2 with tubulin loading control, and densitometry analysis of total SOD2 compared to tubulin. Bottom, a second SDS-PAGE gel probed for acetylated (acetyl-K68) SOD2 with tubulin loading control, and densitometry analysis of acetyl-K68/total SOD2 (from A top) after each were normalized to tubulin loading control.

(B) Western analysis of quadriceps from 11 week-old WT, AR24Q, and AR100Q male mice (n = 3). Endogenous SIRT3 and tubulin loading control with densitometry analysis of SIRT3 normalized to tubulin loading control.

(C) ROS levels in AR112Q expressing PC12 cells (n = 12) treated with 10 nM DHT plus either DMSO (vehicle control), 1 μM viniferin, 1 μM honokiol, or 100 μM melatonin for 48 h (representative image from 3 experiments).

(D) Cell death of AR100Q expressing C2C12 myotubes (n = 3) treated with either DMSO (vehicle control), 1 μM viniferin, 1 μM honokiol, or 100 μM melatonin for 10 days (representative image from 3 experiments).

(E) Left, immunoblot of MYC-tagged SIRT3, and tubulin in AR112Q SIRT3-MYC-overexpressing PC12 cells (n = 3). Right, ROS levels in AR112Q SIRT3-MYC-overexpressing PC12 cells (n = 12) after 48 h of DHT treatment (representative image from 3 experiments).

(F) Left, immunoblot of MYC-tagged SIRT3, and tubulin loading control from AR100Q SIRT3-MYC-overexpressing C2C12 myoblasts (n = 3). Right, percent cell death of AR100Q SIRT3-MYC-overexpressing C2C12 myoblasts (n = 3) (LDH assay) (representative image from 3 experiments). ∗∗ = Nested two-way t-test, p ≤ 0.01.

(A–F) 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) unless otherwise noted. WT = wild type, AR24Q = non-polyQ-expanded AR, AR100Q = polyQ-expanded AR, EV = empty vector, SIRT3 = SIRT3-myc-his overexpression, SIRT3-H248Y = SIRT3-myc-his overexpression (deacetylase inactive).