Additional file 1 of Dysregulation of Tweak and Fn14 in skeletal muscle of spinal muscular atrophy mice

Additional file 1: Supplementary Figure 1. No overt dysregulation of Tweak and Fn14 in skeletal muscle of non-SMA hypomorphic Smn-depleted mice. qPCR analysis of Tweak (a), Fn14 (b), Pgc-1α (c), Mef2d (d), Glut-4 (e), HKII (f) and Klf15 (g) in triceps from post-natal day (P) 7 wild-type (WT), Smn2B/2B and Smn+/- mice. Normalized relative expressions are compared to WT. Data are mean ± SEM, n = 4-5 animals per experimental group, one-way ANOVA, Tukey’s multiple comparison test, * p < 0.05, ns = not significant. Supplementary Figure 2. Tweak and Fn14 are not dysregulated in denervated (nerve cut) muscles of pre-weaned mice. A sciatic nerve cut was performed on post-natal day (P) 7 WT FVB/N mice and both ipsilateral (nerve cut) and contralateral (control) TA muscles were harvested at P14. qPCR analysis of Tweak, Fn14, Pgc-1α, Mef2d, Glut-4, HKII and Klf15 in control and nerve cut TA muscles. Normalized relative expressions for each gene are compared to control muscle. Data are mean ± SEM, n = 7-11 animals per experimental group, two-way ANOVA, uncorrected Fisher’s LSD, ns = not significant. Supplementary Figure 3. Effect of varying Fc-TWEAK doses on disease progression in Smn‍-‍/‍-‍;SMN2 SMA mice. Smn‍-‍/‍-‍;SMN2 mice received daily subcutaneous injections of increasing doses of Fc-TWEAK (7.9, 15., 23.7 and 31.6 μg), starting at birth. a. Daily weights of untreated Smn-/-;SMN2 SMA mice and Smn-/-;SMN2 mice that received daily subcutaneous injections (starting at P0) of Fc-TWEAK (7.9, 15.8, 23.7 and 31.6 μg). Data are mean ± SEM, n = 5-10 animals per experimental group, two-way ANOVA, Sidak’s multiple comparison test. b. Survival curves of untreated Smn-/-;SMN2 SMA mice and Smn-/-;SMN2 mice that received daily subcutaneous injections (starting at P0) of Fc-TWEAK (7.9, 15.8, 23.7 and 31.6 μg). Data are presented as Kaplan-Meier survival curves, n = 5-10 animals per experimental group, Log-rank (Mantel-Cox). Supplementary Figure 4. Differential effect of Fc-TWEAK in skeletal muscle of wild type (WT) and Smn2B/- mice. WT and Smn2B/- SMA mice and received subcutaneous injections of Fc-TWEAK (15.8 μg) every 4 days (from post-natal day (P) 0 to P16) and skeletal muscles were harvested 3 hours post-injections. a-g. qPCR analysis of Fn14 (a), Tweak (b), Pgc-1α (c), Mef2d (d), Glut-4 (e), HKII (f) and Klf15 (g) in triceps of untreated and Fc-TWEAK-treated Smn2B/- mice. Normalized relative expressions for Fc-TWEAK-treated WT mice are compared to untreated WT mice and normalized relative expressions for Fc-TWEAK-treated Smn2B/- mice are compared to untreated Smn2B/- mice. Data are mean ± SEM, n = 3-5 animals per experimental group, two-way ANOVA, uncorrected Fisher’s LSD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. h. Quantification of NF-κB2 p100 protein levels normalized to total protein in the quadriceps of late-symptomatic (P18) Smn2B/- mice and age-matched WT animals. Images are representative immunoblots. Data are mean ± SEM, n = 3-5 animals per experimental group, unpaired t test, p = 0.0005 (WT), p = 0.0494. Supplementary Figure 5. Decreased MuRF-1 expression in skeletal muscle of P7 Fn14-/- mice. qPCR analysis of Atrogin-1 (a) and MuRF-1 (b) in quadriceps and triceps from post-natal day (P) 7 wild type (WT) and Fn14-/- mice. Normalized relative expressions are compared to WT. Data are mean ± SEM, n = 4 animals per experimental group, unpaired t test, p = 0.0164 (MuRF-1 quadriceps), p = 0.0283 (MuRF-1 triceps), ns = not significant.