Fig 1: Effect of Cur, FA, or a combination of both on mitochondrial ROS and Mn-SOD in Aβ1–42-stimulated SH-SY5Y cells. Mitochondrial ROS levels were measured using a mitochondrial ROS detection reagent. Mn-SOD levels were measured using the Human SOD2 ELISA Kit. (A) The levels of mitochondrial ROS in SH-SY5Y cells exposed to 5 µM Aβ1–42 and treated with Aβ1–42 + 1 µM Cur, Aβ1–42 + 10 µM FA or Aβ1–42 + Cur + FA. In the absence of 5 μM Aβ1–42, mitochondrial ROS levels of control, 1 μM Cur, 10 μM FA and Cur + FA-treated cells were 4.26 ± 0.23, 4.38 ± 0.47, 4.52 ± 0.66 and 4.05 ± 0.42 x 104/μg protein (no significant difference, n = 6, Tukey). (B) The concentration of Mn-SOD in SH-SY5Y cells exposed to 5 µM Aβ1–42 and treated with Aβ1–42 + 1 µM Cur, Aβ1–42 + 10 µM FA or Aβ1–42 + Cur + FA. In the absence of 5 μM Aβ1–42, Mn-SOD levels of control, 1 μM Cur, 10 μM FA and Cur + FA-treated cells were 10.83 ± 0.79, 10.47 ± 1.21, 10.23 ± 1.14 and 10.20 ± 0.50 ng/mg protein (no significant difference, n = 6, Tukey). +: inclusion of 5 μM Aβ1–– 42, 1 μM Cur, 10 μM FA, respectively, −: non-inclusion. The p-values in ANOVA were <0.001. Each value expresses the mean + S.E.M. of at least 3 independent experiments. #, p < 0.05; ##, p < 0.01 for Aβ1–42 exposed cells versus the other treated cells (n = 6, Tukey): *, p <0.01 for Aβ1–42 + Cur + FA-treated cells versus Cur-treated cells (n = 6, Tukey).
Fig 2: Secretome analysis of C9 mutated astrocytes. a. Mass spectrometry analysis of control (Contr-L3, Contr-L9) and C9-mutated (C9-L5, C9-L8) astrocyte secretome. 3 biological replicates of media conditioned by each line were analyzed. Conditioned media was collected during toxicity experiments at 70–90 dFD of astrocytes. The results are presented by a volcano plot where each point represents an identified protein. The log2 difference intensity is plotted against the t-test p-value for each protein. The proteins with significantly (p < < 0.05) different levels between C9- and non-mutated secretome samples are in red. b. Proteins related to oxidative stress were significantly down-regulated in the C9-mutated secretome. (c–e) Down-regulation of SOD1, SOD2, and GSS in the C9-mutated secretome was validated by ELISA. 3 biological replicates of conditioned media from C9-mutated (C9-L5, C9-L8) and control (Contr-L3, Contr-L9) astrocytes were analyzed by ELISA assay. Data are presented as a range of values from 3 independent experiments.
Fig 3: Mifamurtide is unable to exert its antitumoral efficacy in osteosarcoma cell lines. Mifamurtide effect on MG--63, HOS and 143-B OS cells in co-culture with macrophages after 24 h of treatment. (A) Representative Annexin V/PI staining plots and relative quantification. (B) FACS analysis and relative quantification of live cells count. (C) Effect of mifamurtide treatment on oxygen reactive species production (ROS) with MitoSOXTM Red assay. Percentage of ROS+ cells are shown. (D) Detection of Intracellular ROS generation using the DCFH-DA assay. Fluorescence intensity of ROS production is shown. (E) Elisa quantification of SOD-2 and (F) Catalase protein expression. Significance was calculated by unpaired Student t-test analysis. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTRL.
Supplier Page from Abcam for Human SOD2 ELISA Kit