Journal: Journal of Ovarian Research

Article Title: Gut microbiota-derived Tryptophanol driven by N-Carbamylglutamate alleviates premature ovarian failure through inhibiting oxidative stress

doi: 10.1186/s13048-026-02152-4

Figure Lengend Snippet: Tryptophanol treatment alleviated POF symptoms by inhibiting PI3K/AKT and MAPK pathways. A Experimental scheme. B Estrus status of mice ( n = 8). C Body weights of mice ( n = 8). D Ovary weights of mice ( n = 8). E Representative Ovary from the Control, the POF and the NCG groups mice with H&E and IHC staining. H&E scale bar = 200 μm; IHC scale bar = 100 μm. F Quantification of primordial, primary, secondary, antral follicles, corpora lutea and corpora atretica per ovary. n = 3 ovaries per each condition. Levels of FSH ( G ), LH ( H ), E 2 ( I ) in mouse serum ( n = 6). Levels of ROS ( J ), MDA ( K ), SOD ( L ), GSH-Px ( M ) in mouse ovaries ( n = 3–4). N Western blotting ( a ) and quantitative ( b ) analysis of proteins ( n = 3). O Litter sizes of mice ( n = 3). Data presented as the mean ± SD, statistical significance was determined using one-way or two-way ANOVA. * p < 0.05, ** p < 0.01. *** p < 0.001. Different lowercase letters indicate significant differences ( p < 0.05)

Article Snippet: To investigate the role of specific signaling pathways, the AKT activator SC79 (MCE, 5 μg/mL) or the MAPK activator Anisomycin (MCE, 10 μM) was added to the culture medium 1 h before downstream assays.

Techniques: Control, Immunohistochemistry, Western Blot

Journal: Journal of Ovarian Research

Article Title: Gut microbiota-derived Tryptophanol driven by N-Carbamylglutamate alleviates premature ovarian failure through inhibiting oxidative stress

doi: 10.1186/s13048-026-02152-4

Figure Lengend Snippet: Tryptophanol treatment alleviated POF by inhibiting PI3K/AKT and MAPK pathways in vitro . A Experimental scheme. B Different concentrations of Cy on GCs viability was assessed by CCK8 assay. C and E Different concentrations of Trp on GCs viability was assessed by CCK8 assay. D The effect of Trp on GCs cell viability following Cy treatment. The control and treatment groups were stained with DCFH-DA, the cells were washed and examined by fluorescence microscopy. Scale bar = 50 μm. F The fluorescence intensity of ROS in mouse GCs. G The level of MDA in mouse GCs. H Flow cytometry apoptosis analysis graph and cell death rate in different groups. The level of SOD ( I ), GSH-Px ( J ), and E 2 ( K ) in mouse GCs. ( L ) Western blotting ( a ) and quantitative ( b ) analysis of proteins ( n = 3). All values are presented as the mean ± SD ( n = 3). Different lowercase letters indicate significant differences compared to the control group ( p < 0.05)

Article Snippet: To investigate the role of specific signaling pathways, the AKT activator SC79 (MCE, 5 μg/mL) or the MAPK activator Anisomycin (MCE, 10 μM) was added to the culture medium 1 h before downstream assays.

Techniques: In Vitro, CCK-8 Assay, Control, Staining, Fluorescence, Microscopy, Flow Cytometry, Western Blot

Journal: Journal of Ovarian Research

Article Title: Gut microbiota-derived Tryptophanol driven by N-Carbamylglutamate alleviates premature ovarian failure through inhibiting oxidative stress

doi: 10.1186/s13048-026-02152-4

Figure Lengend Snippet: Activation of MAPK pathway abrogated the protective effect of Tryptophanol against POF in vitro . A Cell viability of mouse GCs. B Flow cytometry apoptosis analysis graph and cell death rate in different groups. C The level of MDA in mouse GCs. The level of SOD ( D ), GSH-Px ( E ), and E 2 ( F ) in mouse GCs. G Western blotting (a) and quantitative ( b ) analysis of proteins ( n = 3). All values are presented as the mean ± SD ( n = 3). Different lowercase letters indicate significant differences compared to the control group ( p < 0.05)

Article Snippet: To investigate the role of specific signaling pathways, the AKT activator SC79 (MCE, 5 μg/mL) or the MAPK activator Anisomycin (MCE, 10 μM) was added to the culture medium 1 h before downstream assays.

Techniques: Activation Assay, In Vitro, Flow Cytometry, Western Blot, Control

Journal: Scientific Reports

Article Title: Histone acetylation determines transcription of atypical protein kinases in rat neurons

doi: 10.1038/s41598-019-40823-z

Figure Lengend Snippet: Blockade of translation with anisomycin (Ani, 10 μM) selectively altered the TSA-induced regulation of PKMζ and PKCζ expression, while epigenetically-driven upregulation of PKCλ expression was unaffected. ( a ) Application of Ani did not impair the TSA-induced upregulation of PKCλ expression. Control, TSA-treated (19 h) cultures and cultures treated with Ani alone (20 h) or in combinations with TSA were used for comparisons. For the last group, Ani was administered for 1 h before the application of TSA. [F (3,17) = 20,913; p < 0,001; n = 5–6/group]. ( b , c ) Ani prevented downregulation of PKMζ expression in TSA-treated cultures [KW: H (3, N = 24) = 13,22000 p = 0,0042; *p = 0,002; & p < 0,001; n = 6/group] and significantly alleviated epigenetically-driven upregulation of PKCζ expression [KW: H (3, N = 20) = 16,45893 p = 0,0009; *p < 0,001; n = 5/group]. Representative experiment below demonstrates patterns of PKCζ expression in the current experimental series. mRNA levels were determined by standard PCR followed by agarose gel electrophoresis. YWHAZ served as a reference. *Significant differences; & Significant difference (relative to standard control group); ns – not significant.

Article Snippet: Transcription inhibitor actinomycin D (ActD, final concentration 200 nM, 4 μM; Sigma-Aldrich), and protein synthesis inhibitor anisomycin (Ani, final concentration 10 μM, 100 μM; Tocris) were used for verification of experiments with histone deacetylase inhibitors.

Techniques: Expressing, Control, Agarose Gel Electrophoresis

Journal: Kidney International Reports

Article Title: Structure-Activity Analysis Reveals Perturbed Cilia-Jun N-Terminal Kinase Signaling in MAPKBP1-Associated Kidney Disease

doi: 10.1016/j.ekir.2025.05.049

Figure Lengend Snippet: Influence of JNK signaling on MAPKBP1 localization to microtubular structures. (a–c) Immunofluorescence microscopy showing overall (a) intracellular and (b) centrosomal localization of GFP-tagged MAPKBP1 wild type in transiently transfected HeLa cells (GFP-MAPKBP1: green, PCNT: red, nuclei: blue) and localization of GFP-MAPKBP1 wild type at (c) the basal body of transiently transfected H69 cells (GFP-MAPKBP1: green, acetylated-α tubulin: red, nuclei: blue) upon either inhibition (JNK Inh VIII) or activation (anisomycin) of JNK signaling pathway. Scale bars equal (a) 10 μm; (b) 1 μm; (c) 2 μm. (d) Quantification of GFP intensity at the centrosome in HeLa cells transiently transfected with GFP-MAPKBP1 wild type treated with either JNK Inh VIII or anisomycin (AM), respectively. (e) Quantification of GFP intensity at the basal body relative to the cytosol of H69 cells transiently transfected with GFP-MAPKBP1 wild type treated with either JNK Inh VIII or anisomycin (AM), respectively. (f) Quantification of transfected H69 cells presenting a primary cilium (red) and cells with GFP-MAPKBP1 localized at the basal body (green) depending on treatment with either JNK Inh VIII or anisomycin (AM). (g) Quantification of MAPKBP1 wild type in cell lysates of HEK293T cells treated with either DMSO, JNK Inh VIII or anisomycin (AM), respectively. (h) Co immunoprecipitation to quantify homodimerization of MAPKBP1 wild type depending on JNK signaling activation (AM) or inhibition (JNK Inh VIII). (i) Immunofluorescence microscopy of HeLa cells transiently cotransfected with RFP-tagged MAPKBP1 wild type and GFP-tagged MAPKBP1 deletion variants. Images show influence of JNK activation (anisomycin) on homodimerization and cellular localization (RFP-MAPKBP1 wild type: red; GFP-MAPKBP1 variant: green, nuclei: blue). Scale bars equal 10 μm. (j) Quantification of cellular localization pattern depending on JNK signaling activation (anisomycin). All data represent the mean (± SEM) of at least 3 independent experiments. AM, Anisomycin; GFP, green fluorescent protein; JNK Inh VIII/JI VIII, JNK inhibitor VIII; PCNT, pericentrin; RFP, red fluorescent protein; #, ##, reference for statistical testing; ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001; and ∗∗∗∗ P ≤ 0.0001.

Article Snippet: Activation of JNK signaling was induced by incubation with 25 μg/ml anisomycin (AM) (#2222 Cell Signaling, Danvers, MA) or with 10 ng/ml human TGF-beta 1 (# PHG9204, Thermo Fisher Scientific, Waltham, MA) for 1 hour.

Techniques: Immunofluorescence, Microscopy, Transfection, Inhibition, Activation Assay, Immunoprecipitation, Variant Assay

Journal: Kidney International Reports

Article Title: Structure-Activity Analysis Reveals Perturbed Cilia-Jun N-Terminal Kinase Signaling in MAPKBP1-Associated Kidney Disease

doi: 10.1016/j.ekir.2025.05.049

Figure Lengend Snippet: Mutual influence of JNK and MAPKBP1 in RPE1 cells and patient fibroblasts. (a) Immunofluorescence microscopy of RPE1 cells treated with either DMSO, JNK Inh VIII, anisomycin or TGFB. (b) Immunofluorescence microscopy of RPE1 cells transfected with anti- MAPKBP1 siRNA (MAPKBP1-KD) or scrambled siRNA. (a–b I) Endogenous MAPKBP1 was stained with anti-MAPKBP1 antibody (MAPKBP1: red acetylated α-tubulin: green, nuclei: blue). Scale bars equal 2 μm. (a–b II) Quantification of MAPKBP1 intensity at the basal body. (a–b II) Ciliary length. Black bordered white dots represent the mean of each independent experiment. Violin plots represent the sum of all individual datapoints. (c) Immunofluorescence microscopy of RPE1 cells transfected with anti- MAPKBP1 siRNA (MAPKBP1-KD) or scrambled siRNA and treated with either DMSO or TGFB. (I) Endogenous pJNK stained with anti-pJNK antibody (pJNK: red, acetylated α-tubulin: green, nuclei: blue). Scale bars equal 2 μm. (II) Quantification of pJNK intensity at the basal body depending on TGFB treatment. (III) Quantification of nuclear pJNK. (d) Immunofluorescence microscopy of primary fibroblasts of a healthy control individual treated with either DMSO, JNK Inh VIII, anisomycin (AM). (I) Endogenous MAPKBP1 stained with anti-MAPKBP1 antibody (MAPKBP1: red, acetylated α-tubulin: green, nuclei: blue). Scale bars equal 2 μm. (II) Quantification of MAPKBP1 intensity at the basal body. (III) Ciliary length. Black bordered white dots represent the mean of each independent experiment. Violin plots represent the sum of all individual datapoints. (e) Immunofluorescence microscopy of endogenous MAPKBP1 at the basal body of primary cilia in primary fibroblasts from a healthy control individual and a NPHP20 patient. (I) Endogenous MAPKBP1 was stained with primary anti-MAPKBP1 antibody (MAPKBP1: red, acetylated α-tubulin: green, nuclei: blue). Scale bars equal 2 μm. (II) Quantification of pJNK intensities at the basal body in dermal fibroblasts from healthy control individuals I and II, and NPHP20 patients I, II, and V. (III) Quantification of nuclear pJNK in dermal fibroblasts of healthy control I and II and NPHP20 patients I, II, and V. (IV) Ciliary length of primary cells derived from healthy control individuals I and II and NPHP20 patients I, II, and V with and without treatment with 250 nM cytochalasin D. Data are mean ± SEM of the mean values of at least 3 independent experiments. All data represent the mean ± SEM of at least 3 independent experiments. AM, Anisomycin; DMSO, dimethyl sulfoxide; JNK Inh VIII/JI VIII, JNK inhibitor VIII; siRNA, small interfering RNA; TGFB, transforming growth factor beta; #, ##, reference for statistical testing; ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001; and ∗∗∗∗ P ≤ 0.0001.

Article Snippet: Activation of JNK signaling was induced by incubation with 25 μg/ml anisomycin (AM) (#2222 Cell Signaling, Danvers, MA) or with 10 ng/ml human TGF-beta 1 (# PHG9204, Thermo Fisher Scientific, Waltham, MA) for 1 hour.

Techniques: Immunofluorescence, Microscopy, Transfection, Staining, Control, Derivative Assay, Small Interfering RNA

Journal: The American journal of pathology

Article Title: HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via β-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation.

doi: 10.1016/j.ajpath.2016.09.006

Figure Lengend Snippet: Figure 6 Ritonavir up-regulates neuronal BACE1 expression via translation-dependent control. A: Rat neurons were exposed to indicated treatments for 16 hours. BACE1 mRNA was determined using the DDCt method relative to TATA-Box binding protein (TBP). Quantitative RT-PCR assays were performed in triplicate (one-way analysis of variance, Dunnett post hoc). B: Rat neurons were either treated with ritonavir alone or pretreated with anisomycin or acti- nomycin D for 12 hours before ritonavir. Fast green was used as a loading control. C: Compared to ritonavir-only treated neurons, anisomycin pretreatment resulted in decreased BACE1 expression (one-way analysis of variance, Newman-Keuls post hoc). D: Rat neurons were treated for 16 hours, and cytoplasmic and nuclear fractions were analyzed by immunoblotting. E: Densitometric analysis revealed a significant increase in activating transcription factor 4 (ATF4) and BACE1 (one-way analysis of variance, Dunnett post hoc). F: Fourteen DIV rat cortical cultures were transfected with pcDNA3.1Zeoþ vector containing full human BACE1 coding region, including the BACE1 50 UTR but missing the 30 UTR (þ50 UTR), or pcDNA3.1Zeoþ vector containing human BACE1 coding region, including the 30 UTR but missing the 50 UTR (-50 UTR). Forty-eight hours later, cultures were treated with dimethyl sulfoxide or 10 mmol/L ritonavir for 16 hours, and immunoblotted for BACE1. Representative blots are shown. G: Quantification of BACE1 was normalized to actin (one-way analysis of variance, Newman-Keuls post hoc). H: Rat neurons were treated with 10 mmol/L ritonavir, 1 mmol/L saquinavir, or 10 mmol/L lactacystin for 16 hours. Whole cell lysate (10 mg) from each condition was used to determine 20S proteasome activity (one-way analysis of variance, Dunnett post hoc). n Z 4 (A, C, and E); n Z 3 (B); n Z 2 (H). *P < 0.05, **P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UT, untreated.

Article Snippet: The following chemical reagents used in the study were purchased from the indicated vendors: Citifluor, Ltd (London, UK): citifluor AF1; BioRad (Hercules, CA): bradford protein assay dye, polyvinylidene difluoride membrane, and prestained broad-range molecular weight ladder; Jackson ImmunoResearch Labs (West Grove, PA): all dye-conjugated secondary antibodies; Life Technologies (Carlsbad, CA): Dulbecco’s modified Eagle’s medium, neurobasal media, and B27 supplement; Millipore (Temecula, CA): 20S proteasome activity assay; Nanotherics (Newcastle under Lyme, UK): neuromag transfection reagent; Sigma (St. Louis, MO): DAPI, fast green FCF, protease inhibitor cocktail, 4-hydroxytamoxifen (H7904), anisomycin (A9789), and actinomycin D (A1410); Peptides International (Louisville, KY): poly-L-lysine; New England Biolabs (Ipswich, MA): tyramide amplification system; Scytek Labs (Logan, UT): normal antibody diluent; Thermo Fisher Scientific (Waltham, MA): SuperSignal West Dura extended-duration substrate and all horseradish peroxidaseeconjugated secondary antibodies.

Techniques: Expressing, Control, Binding Assay, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Activity Assay