Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs NR4A1 and NR4A2 knockdown on β1-integrin, estrogen receptor β, and mTOR signaling in IHEEC and IHESC cells. IHEEC cells were treated with DIM-3,5 analogs for 24 hours (A) and transfected with siRNAs targeting NR4A1 (siNR4A1) or NR4A2 (siNR4A2) (B) for 72 hours. Whole-cell lysates were then collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 analogs for 24 hours (C) and transfected with siNR4A1 or siNR4A2 for 72 hours (D). Whole-cell lysates were analyzed by Western blots. Protein expression levels were quantified by measuring relative band intensities compared to control siRNA-treated cells (set at 1.0) and normalized to β-actin (A–D). Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; mTOR, mechanistic target of rapamycin; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2; siRNA, small interfering RNA.

Article Snippet: The primary antibodies used for Western blotting were as follows: cleaved poly(ADP-ribose) polymerase (C-PARP) (#5625S, RRID:AB_10699459), β1-integrin (#9699S, RRID:AB_11178800), claudin-1 (#13255T, RRID:AB_2798163), Vimentin (#5741T, RRID:AB_10695459), Slug (#9585T, RRID:AB_2239535), β-catenin (#8480T, RRID:AB_11127855), N-cadherin (#13116T, RRID:AB_2687616), ZO-1 (#8193T, RRID:AB_10898025), ZEB1 (#3396T, RRID:AB_1904164), Snail (#3879T, RRID:AB_2255011), phospho-mechanistic target of rapamycin (mTOR) (#2971S, RRID:AB_330970), mTOR (#2972S, RRID:AB_330978), and epidermal growth factor receptor (EGFR) (#4267T, RRID:AB_2895042), all from Cell Signaling Technology (Danvers, MA, USA); NR4A2 (#sc-376984X, RRID:AB_2893391), TWIST (#sc-81417, RRID:AB_1130910), and estrogen receptor β (ERβ, #sc-8974, RRID:AB_2102246) from Santa Cruz Biotechnology (Dallas, TX, USA); NR4A1 (#ab283264, RRID:AB_3665433) from Abcam (Waltham, MA, USA); β-actin (#A1978, RRID:AB_476692) from Sigma-Aldrich (Milwaukee, WI, USA); and α-smooth muscle actin (α-SMA, #GTX100034, RRID:AB_1240408), collagen type I α1 (COL1A1, #GTX112731, RRID:AB_10721155), connective tissue growth factor (CTGF, #GTX124232, RRID:AB_11169640), and fibronectin (FN, #GTX112794, RRID:AB_1950298) from GeneTex (Irvine, CA, USA); caspase 3 (Cleaved Asp175, #PA5-114687, RRID:AB_2899323) from Thermo Fisher Scientific (Waltham, MA, USA) was used for IHEEC cell line while cleaved caspase 3 (#25128-1-AP, RRID:AB_3073913) from Proteintech (Rosemont, IL, USA) was used for IHESC cell line.

Techniques: Knockdown, Transfection, Western Blot, Expressing, Control, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: The compound ( E )-2-(3,4-dihydroxystyryl)-3-hydroxy-4H-pyran-4-one alleviates neuroinflammation and cognitive impairment in a mouse model of Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-23-01890

Figure Lengend Snippet: Effects of D30 on PI3K-AKT pathway signaling and Gal-3 expression in vitro. (A, B) GO enrichment analysis (bubble map; A) and KEGG pathway analysis (histogram; B) performed as part of the pharmacological network analysis. (C) PI3K, p-AKT, AKT, p-mTOR, and mTOR expression in the hippocampus, as measured by immunoblot, after two fAβ injections. (D) Band densities are expressed as ratios of PI3K, p-AKT, AKT, p-mTOR, and mTOR to β-actin. (E) PI3K , AKT , mTOR , and Gal-3 mRNA expression levels in primary microglial cells stimulated with fAβ, as measured by qPCR. Values are expressed as the mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). AKT: Protein kinase B; D30: ( E )-2-(3,4-dihydroxystyryl)-3-hydroxy-4H-pyran-4-one; fAβ: fibrillar amyloid-β; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; mTOR: mammalian target of rapamycin; p-AKT: phosphorylated protein kinase B; p-mTOR: phosphorylated mammalian target of rapamycin; PI3K: phosphatidylinositol-3-hydroxykinase; qPCR: quantitative polymerase chain reaction.

Article Snippet: Next, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-glial fibrillary acidic protein (GFAP; rabbit, 1:8000, Proteintech, Wuhan, China, Cat# 16825-1-AP, RRID: AB_2109646), anti-Iba1 (goat, 1:1000, Abcam, Cat# ab5076, RRID: AB_2224402), anti-NeuN (rabbit, 1:1000, CST, Danvers, MA, USA, Cat# 24307, RRID: AB_2651140), anti-inducible nitric oxide synthase (iNOS; rabbit, 1:2000, Proteintech, Cat# 22226-1-AP, RRID: AB_2879038), anti-Aβ (rabbit, 1:1000, CST, Cat# 8243, RRID: AB_2797642), anti-phosphatidylinositol-3-hydroxykinase (PI3K; rabbit, 1:1000, CST, Cat# 4249, RRID: AB_2165248), anti-phosphorylated protein kinase B (p- AKT; rabbit, 1:2000, CST, Cat# 4060, RRID: AB_2315049), anti-AKT (rabbit, 1:2000, CST, Cat# 4691, RRID: AB_915783), anti-phosphorylated mammalian target of rapamycin (p-mTOR; rabbit, 1:1000, CST, Cat# 5536, RRID:AB_10691552), anti-mammalian target of rapamycin (mTOR; rabbit, 1:1000, CST, Cat# 2983, RRID: AB_2105622), anti-synaptophysin (rabbit, 1:5000, Abcam, Cat# ab32127, RRID: AB_2286949), anti-postsynaptic density protein 95 (PSD95; mouse, 1:3000, Abcam, Cat# ab13552, RRID: AB_300453), anti-Gal-3 (rabbit, 1:4000, Proteintech, Cat# 14979-1-AP, RRID: AB_2136768), anti-CD68 (rabbit, 1:2000, Proteintech, Cat# 28058-1-AP, RRID: AB_2881049), anti-C3 (rabbit, 1:3000, Abcam, Cat# ab97462, RRID: AB_10679468), anti-TNF-α (rabbit, 1:2000, Proteintech, Cat# 26405-1-AP, RRID: AB_2918102), anti-vesicular γ-aminobutyric acid transporter (VGAT; guinea pig, 1:2000, SYSY, Göttingen, Germany, Cat# 131-004, RRID: AB_887873), anti-β-actin (mouse, 1:6000, Proteintech, Cat# 66009-1-lg, RRID: AB_2782959), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit, 1:1000, CST, Cat# 5174, RRID: AB_10622025).

Techniques: Expressing, In Vitro, Western Blot, Real-time Polymerase Chain Reaction

Journal: World Journal of Gastroenterology

Article Title: Chaperonin-containing tailless complex polypeptide 1 subunit 6A negatively regulates autophagy and protects colorectal cancer cells from cisplatin-induced cytotoxicity

doi: 10.3748/wjg.v31.i18.105729

Figure Lengend Snippet: Chaperonin-containing tailless complex polypeptide 1 subunit 6A affects the autophagy pathway in colorectal cancer cells. A and B: Cell lysates were extracted from knockout (KO)-Control (Ctrl) and KO-chaperonin-containing tailless complex polypeptide 1 subunit 6A (CCT6A) HT29 cells, as well as overexpression (OE)-Ctrl and OE-CCT6A SW480 cells, and then subjected to immunoblotting using the indicated antibodies; C: Following treatment with chloroquine diphosphate salt (CQ, 20 μM, hereafter unless otherwise indicated) for 2 hours, immunoblotting was performed with the total proteins extracted from the indicated cells. DDP: Cisplatin; LC3: Light chain 3; mTOR: Mammalian target of rapamycin; Ulk1: Unc-51-like autophagy activating kinase 1.

Article Snippet: Primary antibodies against phosphorylated mammalian target of rapamycin (phosphor-mTOR) (S2448, 5536S), total-mTOR (2983S), phospho-p70 (T389, 9234S), total-p70 (34475S), total-mixed lineage kinase domain-like (MLKL, 14993S), total-RIP3 (13526S), phospho-RIP1 (S166, 65746S), phospho-RIP3 (S227, 93654S), Bcl-extra large (Bcl-xL) (2764S), poly(ADP-ribose) polymerase 1 (PARP-1, 9542S), and cleaved caspase-3 (9661S) were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Knock-Out, Control, Over Expression, Western Blot