Fig 2: NAC attenuated ERS and apoptosis in PA-treated cardiomyocytes but not in cells treated with high glucose.a Representative western blot analysis of Bcl-2, Bax and cleaved caspase 3 in NRCMs after treatment with HG (30 mM) or PA (300 µM) with or without NAC (1 mM). b–d ERS-associated signaling pathways, such as p-IRE1a/sXBP1 (b), p-PERK/p-eIF2a (c) and GRP78/ATF6/CHOP (d) were also examined by western blotting in NRCMs. GAPDH was used as a loading control. ImageJ software was used to measure the band intensity, and each band intensity was normalized to GAPDH. e Annexin V/PI double staining was performed by flow cytometry to detect apoptosis in NRCMs. All data are presented as the mean±SEM of three independent experiments. *P < 0.05, **P < 0.01 vs control group; #P < 0.05, ##P < 0.01 vs PA-treated NRCMs

Fig 3: NRF2 activation by PERK partially relies on ROS generation during protein synthesis recovery. NCI-H358 cells were treated with AP over a time course of 24 h and RT-qPCR analysis were performed on canonical (a) ATF4-target genes (TRB3, GADD34, CHOP) or (b) NRF2-target genes (NQO1, HO1, GCLC). (c) Time course analysis of the KEAP1/NRF2 complex dissociation following AP treatment. CI-H358 cells were transiently co-transfected with the Neh2-Luc reporter and Renilla vectors and treated with NAC for 1 h prior to the addition of AP for the indicated periods of time. Data were normalized against the Renilla luciferase activity for each condition. Activity fold increase is reported. (d) Western blot analysis of ATF4, NRF2 and CHOP in cytoplasmic and nuclear fractions upon AP-mediated Fv2E-PERK activation with or without NAC. * = non-specific bands. (e) RT-qPCR analysis of NQO1, HO1 and GCLC expression levels. NCI-H358 cells were treated with or without NAC for 1 h prior to the addition of AP for 24 h. (f) RT-qPCR analysis of NQO1, HO1 and GCLC expression levels. NCI-H358 cells were treated with or without NAC for 1 h prior to the addition of tunicamycin for 24 h. Data are expressed as means ± SEM of at least three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: Working model of CHOP-dependent IL-8 regulation, after dabrafenib and trametinib treatment, according to BRAF status.a Upon addition of the BRAF inhibitor dabrafenib, signaling output from BRAFV600E is blocked and there is a transient suppression of ERK activation and MAPK signaling. In this genetic context, CHOP is exported to the cytoplasmic compartment, thus resulting in the downregulation of IL-8. b In contrast, in BRAF-wt contexts, even in the presence of dabrafenib, BRAF forms a complex with CRAF and hyperactivates CRAF itself, thereby driving paradoxical hyper-activation of both MEK and ERK. Due to CHOP nuclear import and histone acetylation, chromatin is accessible to CHOP, thereby increasing its binding to the IL-8 promoter, resulting in IL-8 transcription and protein production. c Trametinib inhibits MEK activity, thereby downregulating p-ERK levels: CHOP translocates from the nucleus to the cytosol and IL-8 gene is not transcribed, regardless of BRAF genetic status.

Fig 5: L2H17 reduces diabetes-induced retinal dysfunction.a Mouse retinal function was determined by electroretinography. Representative electroretinograms (left) and quantification (right) showing the Rod reaction, Max reaction, and Osz potentials (Ops). b Immunofluorescence labeling of 8-OHdG (red), TUNEL (green), and ApoB100 (red) in retinal sections. The number of positive cells (arrow) was counted (Scale bar = 20 µm). c, d The levels of TLR4, NOX4, MyD88, and the ER stress-related protein CHOP in the retinal tissues of mice. GAPDH was used as a loading control. Representative blots are shown in (c), and densitometric quantification is shown in (d). e IL-1ß protein levels in retinal tissue lysates were measured by ELISA. The data were normalized to the total amount of proteins in the samples. Quantitative data are presented as the mean ± SEM, n = 4–8; #P < 0.05; ##P < 0.01; ###P < 0.001 compared to nondiabetic control mice; *P < 0.05; **P < 0.01; ***P < 0.001 compared to untreated diabetic mice.