Fig 1: The unfolded protein response (UPR) and p38 mitogen-activated protein kinase (MAPK) signaling pathways are involved in penfluridol-induced autophagosome accumulation and autophagosome accumulation-mediated cell death is mainly attributed to ATP energy loss.A549 and HCC827 cells were treated with various concentrations of penfluridol for 24 h (a) or 10 μM penfluridol for indicated time points (b), and UPR signals, GRP94, GRP78, ERO1α, PERK, IRE1α, and CHOP, were detected by a western blot analysis. c A549 and HCC827 cells were pretreated with or without cycloheximide (35 μM) for 1 h, followed by penfluridol (7.5 μM) treatment for 24 h. Light chain 3 (LC3) conversion and UPR signals in both cells were detected by a western blot analysis. d Phosphorylation levels of p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase 1/2 (JNK1/2) were assessed using a western blot analysis after treatment of A549 cells with 7.5 μM penfluridol for the indicated time points. e A549 and HCC827 cells were pretreated with or without SB203580 (1 μM) for 1 h followed by penfluridol (7.5 μM) treatment for an additional 24 h. LC3 conversion and UPR signals in both cell lines were detected by a western blot analysis. f A549 cells were treated with indicated concentrations of penfluridol for 24 h and harvested for the detection of intracellular ATP by a Luminescent ATP Detection Assay Kit ab113849 from Abcam (Cambridge, MA). *p < 0.05, ***p < 0.001 compared with the control group. g A549 cells were co-treated with or without ATP (0.1, 0.25, or 0.5 mM) and penfluridol (5 μM) for 24 h; then, the death-inducing effects of penfluridol on cells were determined by counting the colonies formed. Left: representative photomicrographs. Right: data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 compared with the penfluridol treatment only group
Fig 2: Cellular bioenergetics analysis of hAFSCs from normal and fetus-affected gestations. (A) Assessment of cellular energy flux for hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations, shown as a percentage relative to hAFSCs from fetus-unaffected gestations. Comparative measurements were taken using Abcam Extracellular oxygen consumption assay (ab197243) and Abcam Glycolysis assay (ab197244). (B) Analysis of mitochondrial membrane potential (Δψm) of hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations; values are indicated as mean ± SD. Analysis was performed using Abcam TMRE Mitochondrial membrane potential assay kit (ab113852). (C) Assessment of cellular energy content (ATP) of hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations. Measurements were performed using Abcam Luminescent ATP detection assay kit (ab113849). (D–F) ROS production measurement in hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations, performed using Abcam DCFDA Cellular ROS detection assay kit (ab113851). Qualitative evaluation with fluorescence microscopy (D): representative images of ROS production in Normal and Pathology groups (scale bar = 400 μm). Quantitative evaluation with flow cytometry (E,F) representative images of ROS production (E) and median fluorescence intensity evaluation (F) in Normal and Pathology [Pat2] groups. (G) RT-qPCR analysis of genes related to cell metabolism and respiration: HIF1A, NRF1, PPARGC1A, ERRA, PKM, LDHA, PDK1, CAT1, SOD2, and GPX1 in control hAFSCs from normal and fetus-affected gestations. mRNA expression levels were normalized to GAPDH and RPL13A and presented as mean values of ΔCt ± SD. *Denotes significant difference with p < 0.05, as evaluated using Student’s t-test.
Supplier Page from Abcam for Luminescent ATP Detection Assay Kit