Fig 2: Effects of compounds 2, 4, and 18 (at a concentration of 2 µM) on the levels of selected antioxidant enzymes CAT, GPx, HO-1, and SOD-1 and -2, and on the expression of Nrf2 after 6 h of incubation. The THP-1-XBlue-CD14-MD2 cell model was used with quercetin as the standard (2 μM), pyocyanin alone (100 µM) as the positive control (PC), and the vehicle alone as the negative control (NC). ** = p < 0.01; *** = p < 0.001; and **** = p < 0.0001.

Fig 3: Cellular thermal shift assays of acetaminophen binding to NQO2. (a) Schematic presentation of CETSA. The interaction between drug and target protein increases the thermal stability of the protein, which can be measured by separating and quantifying nonaggregated levels of the target protein at different temperatures. (b) SOD1 levels in HeLa cells treated as for CETSA experiments. No significant changes in protein levels were seen in the used temperatures. (c) SOD1 protein levels in cellular thermal shift samples. SOD1 levels did not change significantly when HeLa cells were treated as in CETSA (left panel) or ITDRFCETSA (right panel) experiments. For the ITDRFCETSA experiment the second highest concentration of each chemical was used, as samples reached maximal NQO2 intensity at this concentration. GAPDH was used as a loading control. (d) HeLa cell count and membrane integrity after CETSA treatment. HeLa cells were treated as for CETSA, after which cells were treated with propidium iodide for 15 min before analyzing membrane integrity and cell count using flow cytometer. Values are normalized to samples treated at 37 °C. (e) Cellular thermal shift assay of NQO2. APAP (10 mM), AMAP (10 mM), and quercetin (100 μM) were incubated with HeLa cells for 1 h. Representative NQO2 Western blots of each sample are shown. NQO2 levels were normalized to SOD1 levels used as loading control (not shown). Statistical significances between DMSO and APAP are indicated. Note that the membrane integrity is not maintained at the highest temperatures (panel d), but NQO2 binding is observed already before this, indicating binding in intact cells. APAP and AMAP did not differ significantly. (f) Isothermal (at 72 °C) dose response fingerprints in HeLa cells after 1 h exposure to APAP, nitroacetanilide, resveratrol, and menadione. Representative NQO2 Western blots of each sample are shown. Statistical significance between APAP and nitroacetanilide with 0.5 mM concentration is indicated (N = 3–4 in all panels).

Fig 4: ROS accumulation in DDHD2 KO MEFs.a Non-immortalized WT and DDHD2 KO MEFs were incubated with 2.5 µM CellROX Green for 30 min, and then analyzed by IF microscopy. The graph shows the ratio of the CellROX Green intensity of DDHD2 KO MEFs to that of WT MEFs. b WT MEFs, DDHD2 KO MEFs, and DDHD2 KO MEFs with stable expression of DDHD2-WT-mCherry or DDHD2-S351A-mCherry were incubated with 2.5 µM CellROX Green for 30 min, and then analyzed. The graph shows the ratio of the CellROX Green intensity of DDHD2 KO MEFs or those with stable expression of DDHD2 constructs to that of WT cells. c Lysates (20 µg) of WT and DDHD2 KO MEFs were analyzed by WB with antibodies against SOD1, catalase, and GAPDH. Three different preparations were analyzed. d DDHD2 KO MEFs were transfected with a plasmid encoding FLAG (Empty), FLAG-DDHD2 WT, FLAG-DDHD2-S351A, FLAG-DDHD2-W103R, FLAG-DDHD2-V220F, or FLAG-DDHD2-D660H. At 24 h after transfection, the cells were incubated with 2.5 µM CellROX Green for 30 min and analyzed. The fluorescence intensity of CellROX was quantified and is expressed as the ratio relative to that in WT MEFs. No significant difference in the intensity of CellROX staining was observed between cells transfected with FLAG and SPG-associated mutant constructs. The scale bars for whole cell panels are 10 μm. Data represent means ± SEM for four (a) and three (b and d) independent experiments, respectively. *P < 0.05, Student’s t-test

Fig 5: Hypothalamic atrophy in ALS patients. a Representative T1w MRI of hypothalami of a healthy control and an ALS patient; hypothalami are shadowed in red (arrows). b The hypothalamic volume was significantly decreased in ALS patients (n = 72) compared to the controls (n = 43). Bars represent mean ± SD. ****P < 0.000 1