DDIT3/CHOP Antibody - #AF6277

Figure 7. Effect of A22 on ameliorating apoptosis, ER stress, inflammation, metabolic syndrome, and fibrogenesis in HF diet-fed mice. (A) Effect of A22 on BCL-2 gene transcription. (B) Effect of A22 on BAX gene transcription. (C) Effect of A22 on expressions of apoptosis-related proteins in liver. The extracted proteins from the liver were immunoblotted with specific antibodies, and quantified based on the loading control of ACTIN. (D) Effect of A22 on ER stress. The UPR proteins (IRE-1, PERK, elF-2 and CHOP) were analyzed by using western Blot. (E) Effect of A22 on expressions of inflammatory factors. (F) Effect of A22 on expressions of fibrogenic proteins.

Figure 2. A. muciniphila increased markers related to lipid metabolism in liver and gut of HFC mice. HFC induced obese MAFLD (11 weeks of feeding) were administered with saline as the control or A. muciniphila (6 weeks of treatment) to assess liver and ileum parameters: a-c for parameters in the liver and d-f for parameters in the ileum. e-g, representative images of the ileum (Scale bar, for 100 µm). (a) Hepatic mitochondrial copy number determination. (b) Gene expression related to lipid uptake and oxidation in the liver of mice. (c) Protein levels of metabolic regulators mitochondrial complexes, and the LKB1-AMPK axis in the liver of mice. The protein levels were quantified and normalized to the loading control actin (d) Gene expression of lipid uptake and oxidation in the ileum of mice. The gene level or protein level in the HFC group was set as 1, and the relative fold increases were determined by comparison with the HFC control group. (e) Immunohistochemistry analysis of PGC-1α in the ileum of mice. The brown dot indicates the examined protein. Representative images were captured. Scale bar, 100 µm. (f) TG level quantification (indicated by oil red O staining) and oxidative stress-induced cell apoptosis determination (indicated by CHOP examination) in the ileum. Representative images were captured. Scale bar, 100 µm. (g) Immunohistochemistry analysis of E-cadherin and H

Fig. 5 Efect of erianin and E/DMSNs on cytosolic calcium levels and ERS signaling pathway. a Flow cytometry analysis of cytosolic calcium levels in HaCaT cells after treatment with erianin and E/DMSNs for 24 h. b Relative MFI of control in (a) analyzed by fow cytometry. c The expressions of PERK, ATF6, IRE1α, and CHOP proteins after treatment with erianin and E/DMSNs for 24 h. d Quantitation of PERK, ATF6, IRE1α, and CHOP proteins normalized to β-actin in (c) by using Image J software. Values are represented as means±SD (n=3). *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, signifcantly diferent compared with the erianin group. ##p<0.01, ###p<0.005, ####p<0.001, signifcantly diferent compared with the control group

Fig. 5 Effect of erianin and E/DMSNs on cytosolic calcium levels and ERS signaling pathway. a Flow cytometry analysis of cytosolic calcium levels in HaCaT cells after treatment with erianin and E/DMSNs for 24 h. b Relative MFI of control in (a) analyzed by flow cytometry. c The expressions of PERK, ATF6, IRE1α, and CHOP proteins after treatment with erianin and E/DMSNs for 24 h. d Quantitation of PERK, ATF6, IRE1α, and CHOP proteins normalized to β-actin in (c) by using Image J software. Values are represented as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, significantly different compared with the erianin group. ##p < 0.01, ###p < 0.005, ####p < 0.001, significantly different compared with the control group

Fig. 5. Exo-Que inhibits ER stress and ferroptosis in scleral fibroblasts during myopia progression. (A, B) Morphological changes of scleral ER and mitochondria as detected by transmission electron microscopy in different groups at 2 weeks and 4 weeks (n = 3). Blue arrows indicate the ER, green arrows indicate the mitochondria, and red arrows indicate disruptions of the ER or mitochondria; (C) Quantitative analysis of iron content, MDA level, and GSH level (n = 3); (D–G) Expression levels of ER stress- and ferroptosis-related proteins GRP78, PERK, eIF2, p-eIF2, IRE1, XBP1, ATF6, CHOP, ACSL4, and GPX4 in sclera tissues at 2 and 4 weeks (n = 3 at each time point), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; (H and I) The mRNA levels of GRP78, PERK, eIF2, IRE1, XBP1, ATF6, CHOP, ACSL4, and GPX4 were measured by qPCR in the sclera tissues at 2 and 4 weeks (n = 3 at each time point).∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, p-values represent the statistical comparisons between different groups and the FDM group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3: MSR signature enhanced by NAD supplementation in multiple cell models. a–j Levels of UPRmt-related chaperone and protease proteins in N2a APPswe cells (n = 6 per group). At least three experiments were repeated independently with similar results. Data were analyzed by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. k Confocal images of HEK293 Tau P301L cells double immunostained with ATF4 (green) and mitochondria outer membrane protein TOM20 (red) antibody, after 24 h of NMN incubation. Scale bar, 10 μm. l–n 3D reconstruction images of HEK293 Tau P301L cells double immunostained with CHOP (green) and CYC (red) antibodies. Scale bar, 10 μm. o Relative levels of proteins implicated in the mitophagy pathway. p Quantification of the colocalization of the mitochondrial protein CYC and the autophagy protein LC3 in HEK293 Tau P301L cells after treatment with NMN. Scale bar, 10 μm. Immunoblot data were analyzed by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test while the PCC were analyzed by two-sided unpaired t-test. q UPRmt transcript levels in N2a neuroblastoma cell line. All experiments were performed independently with at least three biological replicates. Data are shown as mean ± s.e.m. The p-values are indicated on the graphs. ns not significant.

Fig. 4: Nelfinavir (NFV) activates ER stress. A Western blot analyses for ER stress related protein GRP78, ATF4, CHOP, and IRE1α in Hepa1-6 (n = 3) and HepG2 (n = 3) cells after 24 h treatment of NFV (40 µM). B Effect of TUDCA (200 μM) on NFV-induced upregulation of ER stress associated protein of GRP78 and CHOP, and autophagy associated protein LC3 after 24 h treatment. n = 3 independent replicates for Hepa1-6 and HepG2 cells respectively. C Effect of TUDCA on NFV-induced cell death after 48 h treatment. n = 3 independent replicates for Hepa1-6 and HepG2 cells respectively. D Effect of TUDCA on NFV-induced lipid peroxidation, quantified by MDA level after 24 h treatment. n = 3 independent replicates for Hepa1-6 and HepG2 cells respectively. E, F Effect of TUDCA on NFV-induced lipid peroxidation, quantified by C11-BODIPY fluorescence probe after 24 h treatment. n = 3 independent replicates for Hepa1-6 and HepG2 cells respectively. Scale bar=100 μm. G Effect of TUDCA on NFV-mediated downregulation of SLC7A11, GPX4, Ferritin protein expression after 24 h treatment. n = 3 independent replicates for Hepa1-6 and HepG2 cells respectively. H Effect of TUDCA on NFV-mediated decrease of GSH level after 24 h treatment. n = 3 independent replicates for Hepa1-6 and HepG2 cells respectively. Data were presented as Mean ± SD. Differences were assessed by one-way ANOVA followed by Tukey’s tests. **p 

Fig. 1. Serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) knockdown (3cKD) in adipocytes enhanced endoplasmic reticulum overoxidation and endoplasmic reticulum stress (ERS), and promoted the expression of pro-inflammatory cytokines and adipocyte apoptosis under lipotoxicity injury. (A) Western blotting of Serpina3c protein level in 3cKD 3T3-L1 adipocytes and its control LV3 group, as well as Serpina3c overexpression (3cOV) 3T3-L1 adipocytes and its control LV5 group. (B) Cell counting kit-8 (CCK8) assay measured the cell viability (%) of LV3 group and 3cKD group after 500 μM palmitic acid (PA)treated for 24 or 48 hours. (C-J) In these experiments, LV3, 3cKD, LV5, and 3cOV groups were treated by 500 μM PA for 48 hours. (C, D) The relative mRNA levels of indicated genes in 3T3-L1 adipocytes. (E) The protein levels of indicated genes in 3T3L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (F) Hydrogen peroxide (H2O2) level in 3T3-L1 adipocytes was determined. (G) The protein levels of ERS makers in 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (H) Reactive oxygen species (ROS) level in 3T3-L1 adipocytes was detected. (I) The protein levels of mitogen-activated protein kinase signaling pathway in 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (J) Caspase-3 activity in 3T3-L1 adipocytes was determined. Data were presented as mean±standard error of the mean (n=5 for each group unless otherwise mentioned). NS, no significance; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-X-C motif chemokine ligand 1 or 10; HIF1α, hypoxia-inducible factor 1α; Ero1α, endoplasmic reticulum oxidoreductase 1α; PDIA3 or PDIA4, protein disulfide isomerase family A member 3 or 4; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein; p-eIF2α, phosphorylated eukaryotic initiation factor 2α; c-ATF6, cleaved activating transcription factor 6; XBP1S, spliced X-box binding protein 1; p-JNK, phosphorylated c-Jun N-terminal kinase; p-ERK, phosphorylated extracellular signal-regulated kinase.