Fig 2: Imbalance in ROS-Glutathione Homeostasis that Blunts the DNA Damage Response by Persistent Oxidative Phosphorylation in A-iPSC and Recovery by GLUT3 Expression(A) Excessive oxidation capacity with elevated glutathione in A-iPSC and recovery by GLUT3. Glutathione analysis was conducted with the glutathione fluorometric assay. Error bars indicate SEM of three biological replicates with two independent clones in each sample group. Statistical significance was determined by two-sided t test.(B) A cellular reactive oxygen species assay kit (Abcam, ab113851) was used to measure the H2O2-scavenging activity of ESC, Y-iPSC, A-iPSC, and A-iPSC-GLUT3. Error bars indicate SEM of three biological replicates with two independent clones in each sample group. Statistical significance was determined by two-sided t test.(C) Recovery of ATM phosphorylation in A-iPSC-GLUT3 compared to A-iPSC after phleomycin treatment (2 hr, 30 µg/mL), as monitored by immunoblot in three independent representative clones.(D) In situ cell death assays of Y-iPSC, A-iPSC, Y-iPSC-shGLUT3, and A-iPSC-GLUT3 were performed 15 hr after the end of phleomycin treatment (2 hr, 30 µg/mL). Y-iPSC-shGLUT3 shows fewer cells staining for cell death compared to Y-iPSC and A-iPSC-GLUT3. The negative control is Y-iPSC treated with dye in the absence of enzymatic reaction. Nuclei are stained with DAPI. Scale bar indicates 100 µm.(E) Quantification by image analysis of apoptotic response by DNA fragmentation assay after phleomycin treatment. Error bars indicate SEM of six biological replicates with three independent clones in each sample group. Statistical significance was determined by unpaired two-sided t test.(F) Glucose uptake in Y-iPSC-shGLUT3 is reduced compared to Y-iPSC. Error bars indicate SEM of three biological replications in each sample group and two additional clones of Y-iPSC-shGLUT3. Statistical significance was determined by two-sided t test.(G) The H2O2-scavenging activity of Y-iPSC and Y-iPSC-shGLUT3. Error bars indicate SEM of five biological replicates in each sample group and three additional independent clones of Y-iPSC-shGLUT3. Statistical significance was determined by two-sided t test.(H) Excessive oxidation capacity with elevated glutathione in Y-iPSC-shGLUT3 compared with Y-iPSC. The total glutathione level was measured to determine the maximum oxidation capacity. Glutathione analysis was conducted with the glutathione fluorometric assay. Error bars indicate SEM of five biological replicates in each sample group and two additional clones of YiPSC-shGLUT3. Statistical significance was determined by two-sided t test.(I) Excessive oxidation capacity with elevated glutathione in Y-iPSC-shGLUT3. Glutathione analysis was conducted with the glutathione fluorometric assay. Error bars indicate SEM of three biological replicates with two independent clones in each sample group. Statistical significance was determined by two-sided t test.

Fig 3: Effects of BPE on poly (I:C)-induced pro-inflammatory mediators in RAW 264.7, A549, and WI-38 cells. (A) Raw 264.7 cells were pretreated with different concentrations of BPE or PBS (Control, CTL) for 1 h and then stimulated with poly (I:C) (10 μg/mL) for 6 h. The mRNA levels of IL-6 and IL-10 were measured by real-time reverse-transcription polymerase chain reaction assay and normalized to 18S mRNA level. (B) A549 and WI-38 cells were pretreated with different concentrations of BPE, 10 μM of PDTC or PBS (Control, CTL) for 1 h and then stimulated with poly (I:C) (10 μg/mL) for 6 h. The mRNA level of IL-6 was measured by real-time reverse-transcription polymerase chain reaction assay and normalized to GAPDH mRNA level. (C) A549 and WI-38 cells were pretreated with different concentrations of BPE or PBS (Control, CTL) for 1 h then incubated with poly(I:C) (10 μg/mL) for 24 h. The amount of nitric oxide (NO) released into the media was measured by Griess reagent. (D) The cellular reactive oxygen species (ROS) levels of the cells described in (C) were measured by DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851, Abcam). The values represent mean ± SD (n=3). #p < 0.01, compared with control (CTL) group. *p < 0.05; **p < 0.01; ***p < 0.001, compared with poly (I:C) alone-treated group.

Fig 4: CBD treatment alters markers of mitochondrial and cellular stress, antioxidant capacity and differentiation, and induces oxidative stress in syncytiotrophoblasts. Differentiated (ST) BeWo b30 cells were treated with 20 μM CBD over 48 h. (A–D) Changes in mRNA levels of HSP60, HSP70, SOD1 and SOD2 were normalized to 18S and β-Actin and were assessed using RT-qPCR (N ≥ 5 biological replicates), relative to vehicle control (Veh). (E,F) Changes in protein levels of 4HNE (n = 4 biological replicates) were assessed using Western blotting and normalized to β-actin expression. Cell lysates (25 μg/lane) of each treatment were loaded on SDS-PAGE and probed using a rabbit polyclonal anti-4HNE (Abcam, ab46545) antibody. (G) Intracellular ROS levels were quantified using the DCFDA assay (Abcam, ab113851) in ST cells following treatment with 20 μM CBD compared to vehicle control (n = 24 biological replicates). A total of 10 nM of rotenone (Rot) was used as a positive control and results were normalized to total protein content determined through the BCA assay. (H) Mitochondrial membrane potential (ΔΨm) was determined in both untreated (Utx) cells and following 0 (vehicle), 1, 10 and 20 µM of CBD treatment (n = 6 biological replicates per treatment condition) in STs using the JC-1 assay kit (Abcam, ab113850). ST vehicle = 0.1% methanol, EGF, FSK. Results were plotted as mean ± SEM and compared using either Student’s t-test (for groups ≤ 2), or one-way ANOVA (for groups ≥ 3): p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****). Statistically significant changes were represented by distinct letters on bar graphs where any different letter represents a significant difference of at least p < 0.05.

Fig 5: Calpain inhibition decreases ROS production and cell death in VSC4.1 motoneuron cells. (A) VSC4.1 cells were treated with either 40 ng/mL IFN-γ or 40 ng/mL IFN-γ + 10 µM CP overnight. ROS assay was performed in 96-well plate by the Reactive Oxygen Species Assay Kit (ab113851) (Abcam, Cambridge, UK). IFN-γ induced production of ROS when the cells were treated overnight. Calpain inhibition reduced ROS production in VSC4.1 cells when the cells were treated with CP along with IFN-γ. Data are representative of three separate experiments. (B) VSC4.1 cells treated overnight with IFN-γ (40 ng/mL) or IFN-γ + CP (10 µM) were tested for cell survival by the MTS assay. IFN-γ treatment produced a significant reduction in cell survival, whereas calpain inhibition by CP increased cell viability. ns = no significant difference.