Torkinib

Stomata in the plant epidermis open in response to light, but the molecular mechanisms underlying this process remain incompletely understood. In this study, we screened for compounds that affect light-induced stomatal opening and identified two stomatal closing compounds (SCLs), SCL10 and SCL11 (clorgyline). Both compounds completely suppressed light-induced stomatal opening in Commelina benghalensis but did not affect fungal toxin fusicoccin (FC)-induced opening. The half-maximal inhibitory concentrations (IC50) of SCL10 and SCL11 on light-induced opening were 1.40 and 38.25 μM, respectively. Consistently, both compounds fully inhibited blue-light-induced phosphorylation of the penultimate residue, a threonine (Thr948; numbering according to Arabidopsis AHA1) of plasma membrane (PM) H+-ATPase, which is essential for stomatal opening, but did not interfere with FC-induced phosphorylation in Arabidopsis thaliana guard cells. Notably, red- and blue-light-induced phosphorylation of Thr881 in PM H+-ATPase, another key step in stomatal opening, was inhibited by SCL11 but not by SCL10 in guard cells. Additionally, stomatal opening induced by PP242, a compound that promotes stomatal opening partly by suppressing steady-state abscisic acid signaling, was inhibited by SCL11 but not by SCL10. These results imply that SCL10 inhibits blue-light-induced phosphorylation of Thr948 and stomatal opening upstream of the site of action of PP242, whereas SCL11 inhibits the phosphorylation of both Thr948 and Thr881, and acts downstream of the site of action of PP242. Finally, the compounds were sprayed onto rose leaves, and we observed that wilting was suppressed only in leaves sprayed with SCL10, for up to 5 h.

Objective Exploring the role of transcription factor EB (TFEB)-mediated nuclear translocation in lysosomal degradation of glutathione peroxidase 4 (GPX4) in ferroptosis of human embryonic trophoblast cells HTR8-S/Vneo. Methods HTR8-S/Vneo cells were divided into the following groups: normal group, hypoxia group, hypoxia+si-NC group (cells transfected with si-NC), hypoxia+si-TFEB group (cells transfected with si-TFEB), hypoxia+si-TFEB+RSL3 group (cells transfected with si-TFEB and treated with the ferroptosis inducer RSL3), and hypoxia+si-TFEB+PP242 group (cells transfected with si-TFEB and treated with the lysosome activator PP242). The viability, invasion number, and migration rate of cells were assessed by the CCK-8 kit, Transwell assay, and wound healing assay, respectively. The content of Fe²⁺ in cells was detected using the FerroOrange probe. ROS levels were measured using the DCFH-DA reactive oxygen species (ROS) fluorescent probe. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and the activity of superoxide dismutase (SOD). Western blot analysis was performed to examine the protein expression of TFEB, phosphorylated TFEB (p-TFEB), GPX4, and TFEB nuclear translocation. The lysosomal fluorescent intensity in cells was assessed using a lysosomal green fluorescent probe. Results Compared with the normoxia group, the hypoxia group exhibited significantly reduced viability, invasion number, and migration rate of cells. Intracellular Fe²⁺ levels, ROS fluorescence intensity, LDH release, and MDA level were markedly increased, while SOD activity and GSH levels were significantly decreased. TFEB and p-TFEB protein expression showed significant up-regulation, whereas GPX4 protein expression was notably down-regulated. TFEB nuclear translocation occurred with enhanced lysosomal fluorescence intensity. Compared with the hypoxia+si-NC group, the hypoxia+si-TFEB group demonstrated significantly increased viability, invasion number, and migration rate of cells, accompanied by reduced ferroptosis levels. TFEB nuclear translocation was inhibited, and lysosomal fluorescence intensity decreased. Compared with the hypoxia+si-TFEB group, ferroptosis-related indicators in HTR8-S/Vneo cells were reversed in the hypoxia+si-TFEB+RSL3 group. In contrast, the cellular ferroptosis level was elevated in the hypoxia+si-TFEB+PP242 group. Conclusion Inhibition of TFEB nuclear translocation reduces ferroptosis in human embryonic trophoblast cells HTR8-S/Vneo, possibly by suppressing the lysosomal degradation of GPX4.