Fig. 4

Niclosamide induces phenotypic changes in ALS fibroblasts. Fibroblasts from ALS patients with no known ALS variants (uvALS) and C9orf72 ALS patients were treated for 72 h with three different concentration of niclosamide (Nic): 1 μM, 5 μM, and 10 μM. a Protein expression levels of uvALS fibroblasts were analyzed by western blot with anti-S100A4, anti-p-STAT3, anti-p-mTOR, anti-α-SMA, and anti-N-cadherin. GAPDH was used as a loading control. Data represent mean ± SEM. n = 3 individuals, experiments repeated in triplicate. One-way ANOVA with Tukey correction between multiple comparisons. F value (DFn, DFd): (3,8) = 8.053 (S100A4), (3,8) = 83.52 (p-STAT3), (3,8) = 6.182 (p-MTOR), (3,8) = 9.100 (α-SMA), (3,8) = 7.665 (N-cadherin). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. untreated (Ctrl) cells. b Protein expression levels of C9orf72 fibroblasts were analyzed by Western blot with anti-S100A4, anti-p-STAT3, anti-p-mTOR, anti-α-SMA, and anti-N-cadherin. GAPDH was used as a loading control. Data represent mean ± SEM. n = 3 individuals, experiments repeated in duplicate. One-way ANOVA with Tukey correction between multiple comparisons. F value (DFn, DFd): (3,8) = 6.264 (S100A4), (3,8) = 63.13 (p-STAT3), (3,8) = 7.163 (p-MTOR), (3,8) = 19.08 (α-SMA), (3,8) = 6.757 (N-cadherin). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. untreated (Ctrl) cells. c mRNA levels of S100A4 and α-SMA in uvALS fibroblasts treated with 10 μM niclosamide for 72 h. Data are normalized to GAPDH. Values are mean ± SEM, n = 3 individuals, experiments repeated in triplicate; two-tailed t test. t value: 9.961, degrees of freedom: 4 (S100A4); t value: 3.331, degrees of freedom: 4 (α-SMA). *p < 0.05 and **p < 0.001 vs. untreated (Ctrl) cells. d mRNA levels of S100A4 and α-SMA in C9orf72 fibroblasts treated with 10 μM niclosamide for 72 h. Data are normalized to GAPDH. Values are mean ± SEM, n = 3 individuals, experiments repeated in triplicate; two-tailed t test with Welch’s correction. t value: 14.52, degrees of freedom: 2.094 (S100A4); two-tailed t test. t value: 14.16, degrees of freedom: 4 (α-SMA). *p < 0.01 and **p < 0.001 vs. untreated (Ctrl) cells