am80  (Tocris)

Journal: NPJ Breast Cancer

Article Title: Effects of RARα ligand binding domain mutations on breast fibroepithelial tumor function and signaling

doi: 10.1038/s41523-024-00716-5

Figure Lengend Snippet: Mammalian two- hybrid assay of interactions of the wild type or mutant RARα LBD with a fragment of coactivator SRC2, b MED1, a component of the transcription pre-initiation complex and c fragment of corepressor NCOR, treated with increasing concentrations of RA. Left panel: Partially activatable mutants, Right panel: Non-activatable mutants. N = 3, Error bar = SD. d Binding affinities of WT or mutant RARα LBD with fragment of coactivator SRC1 (SRC1-NR2) in the presence of RA and Am80. N = 3, error bar = SE. **p < 0.01.

Article Snippet: Compounds tested include all-trans retinoic acid (known as RA throughout the manuscript, Sigma R2625), Am80 (aka Tamibarotene, Tocris 3507), AM580 (Tocris 0760) and Ch55 (Tocris 2020).

Techniques: Two Hybrid Assay, Mutagenesis, Binding Assay

Journal: NPJ Breast Cancer

Article Title: Effects of RARα ligand binding domain mutations on breast fibroepithelial tumor function and signaling

doi: 10.1038/s41523-024-00716-5

Figure Lengend Snippet: a Overexpression of Flag-tagged RARα WT and mutants in PT024 cells. b Cell viabilities of PT024 WT or mutant RARα expressing cells after treatment with increasing concentration of RA or Am80. N = 3, error bar = SD. *** p < 0.001, **** p < 0.0001. c Cell cycle distribution of PT024 WT or mutant RARα expressing cells when treated with either vehicle or 100 nM Am80. N = 2, error bar = SD. * p < 0.05, **** p < 0.0001. d GSEA analysis shows downregulation of retinoic acid signaling gene targets in mutant RARα-expressing cell lines. e Heatmap of genes associated with reactome retinoic acid signaling pathway. f Quantitative PCR of retinoic acid signaling associated genes. N = 3, error bar = SD. * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001.

Article Snippet: Compounds tested include all-trans retinoic acid (known as RA throughout the manuscript, Sigma R2625), Am80 (aka Tamibarotene, Tocris 3507), AM580 (Tocris 0760) and Ch55 (Tocris 2020).

Techniques: Over Expression, Mutagenesis, Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Stiff extracellular matrix activates the transcription factor ATF5 to promote the proliferation of cancer cells

doi: 10.1016/j.isci.2025.112057

Figure Lengend Snippet: ATF5 is highly localized in the nuclei of human and mouse pancreatic cancer cells in stiff tumors (A) Representative staining images of hematoxylin and eosin (HE) and immunohistochemistry for ATF5 and EGR1 in adjacent normal, and collagen-poor and collagen-rich tumor regions of human pancreatic ductal adenocarcinoma. The areas of black rectangles are magnified. (B) Proportions of cells negative for ATF5 (−), nuclear positive for ATF5 (Nuc(+)), and nuclear negative and cytoplasm-positive for ATF5 (Other(+)) in human pancreatic cancer tissues ( n = 9 patients) were examined via immunohistochemistry, followed by quantification. (C) Proportions of cells negative for EGR1 (−), nuclear-positive for EGR1 (Nuc(+)), and nuclear-negative and cytoplasm-positive for EGR1 (Other(+)) in human pancreatic cancer tissues ( n = 9 patients) were examined by immunohistochemistry, followed by quantification. (D) Representative staining images of hematoxylin and eosin (HE) and immunohistochemistry for ATF5 and EGR1 in control pancreatic tumors and those treated with AM80. The areas of black rectangles are magnified. Note that AM80 is reported to reduce tumor stiffness by inducing changes in the phenotype of CAFs. (E) Proportions of cells negative for ATF5 (−), nuclear positive for ATF5 (Nuc(+)), and nuclear negative and cytoplasm-positive for ATF5 (Other(+)) in tumor tissues obtained from control mice ( n = 9) and those administered AM80 ( n = 10) were examined via immunohistochemistry followed by quantification. (F) Proportions of cells negative for EGR1 (−), nuclear positive for EGR1 (Nuc(+)), and nuclear negative and cytoplasm-positive for EGR1 (Other(+)) on tumor tissues obtained from control mice ( n = 9) and those administered AM80 ( n = 10) were examined via immunohistochemistry followed by quantification. Scale bar = 100 μm; mean with S.D. and each data point is shown; †, statistical significance ( p < 0.05) determined via Student’s t test; ‡, statistical significance ( p < 0.05) determined via Welch’s t-test; ¥, statistical significance ( p < 0.05) determined via Wilcoxon rank-sum test; §, statistical significance ( p < 0.05) determined via paired t-test. For multiple comparisons, we analyzed significance using the Bonferroni correction.

Article Snippet: AM80 , Tocris , Cat# 3507.

Techniques: Staining, Immunohistochemistry, Control

Journal: iScience

Article Title: Stiff extracellular matrix activates the transcription factor ATF5 to promote the proliferation of cancer cells

doi: 10.1016/j.isci.2025.112057

Figure Lengend Snippet:

Article Snippet: AM80 , Tocris , Cat# 3507.

Techniques: Purification, Recombinant, Plasmid Preparation, Modification, Cloning, Transfection, In Vitro, Isolation, Staining, Control, Western Blot, Microarray, shRNA, Software

Journal: Life Science Alliance

Article Title: NAD + depletion is central to placental dysfunction in an inflammatory subclass of preeclampsia

doi: 10.26508/lsa.202302505

Figure Lengend Snippet: (A, B, C) Gene expression of inflammatory markers in human placenta biopsies from all three PE subclasses (n = 4 placentas/group). (B, C) Representative immunofluorescence images and quantification of protein ADP-ribosylation in trophoblasts of human placental tissue sections from all three PE subclasses and various control groups (n = 7–8 placentas/group). ADP-ribosylation was measured specifically within the trophoblasts highlighted using the white lines. (D, E, F, G) LC/MS quantification of human placental NAD(H) levels (n = 15–42 placentas/group). (H) Heatmap of down-regulated mitochondrial proteins in inflammatory PE3 (term) placentas compared with control term placentas (n = 5–6 per group). Shown are only proteins that met the false discovery rate threshold of 0.05. (I, J) Placental oxidative stress was determined by immunofluorescence staining of human placental tissue sections with anti-8-oxo-dG antibody and quantification by the integrated intensity/area of trophoblasts (n = 4 per group). One-way ANOVA with Holm–Šídák’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001. The error bar indicates the SD. Control = control term and preterm, CH = chronic hypertension control, PE1 = gestational parent–driven PE subclass, PE2 = hypoxia-driven PE subclass, PE3 = inflammation-driven PE subclass.

Article Snippet: The membranes were then incubated with primary anti-ADP-ribosylation antibody (AM80-100UG; MilliporeSigma) diluted in TBS-T buffer overnight at 4°C with gentle rocking.

Techniques: Gene Expression, Immunofluorescence, Control, Liquid Chromatography with Mass Spectroscopy, Staining

Journal: Life Science Alliance

Article Title: NAD + depletion is central to placental dysfunction in an inflammatory subclass of preeclampsia

doi: 10.26508/lsa.202302505

Figure Lengend Snippet: (A, B) Trophoblast protein ADP-ribosylation was determined by Western blot with anti-ADP-ribosylation antibody on cells treated with 10 ng/ml TNF-α and/or 150 μM–1 mM NR for 24 h (n = 3). One-way ANOVA with Holm–Šídák's multiple comparisons test. (C) Total cellular NAD + (H) and NAD + /NADH ratio quantification was performed on cells treated with or without 10 ng/ml TNF-α for 24 h (n = 3). One-tailed unpaired t test. (D, E, F, G) XFe96 Seahorse Mito stress test was performed on cells treated with 10 ng/ml TNF-α and/or 250 μM NR for 24 h. Basal, ATP-linked, and maximal mitochondrial respiration rates and extracellular acidification rates were measured (n = 4). (H, I, J, K, L) Expression of oxidative phosphorylation (OXPHOS) proteins was determined by Western blot with OXPHOS antibody cocktail on cells treated with 10 ng/ml TNF-α and/or 250 μM NR for 24 h (n = 4–7). (M, N) Trophoblast invasion capacity was determined by performing a Matrigel invasion assay on cells treated with 10 ng/ml TNF-α and/or 250 μM NR for 72 h (n = 3). Two-way ANOVA with Holm–Šídák's multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001. The error bar indicates the SD. TNF-α = tumor necrosis factor, NR = nicotinamide riboside. Source data are available for this figure.

Article Snippet: The membranes were then incubated with primary anti-ADP-ribosylation antibody (AM80-100UG; MilliporeSigma) diluted in TBS-T buffer overnight at 4°C with gentle rocking.

Techniques: Western Blot, One-tailed Test, Expressing, Phospho-proteomics, Invasion Assay

Journal: Life Science Alliance

Article Title: NAD + depletion is central to placental dysfunction in an inflammatory subclass of preeclampsia

doi: 10.26508/lsa.202302505

Figure Lengend Snippet: (A) Placental TNF-α levels were determined by ELISA (n = 7–9 litters/treatment, 1 placenta/litter). (B, C) Placental protein ADP-ribosylation was determined by Western blot with anti-ADP-ribosylation antibody (n = 10 litters/treatment, 1 placenta/litter). (D) Placental ADP-ribose (ADPR) levels were determined by LC/MS (n = 6–10 litters/treatment, 1 placenta/litter). (E, F, G, H) Placental NAD + (H) levels were determined by a colorimetric assay (n = 8–10 litters/treatment, 1 placenta/litter). (I, J) Placental mitochondrial function was determined by performing an oxygraph assay on isolated mitochondria. Complex I– and complex II–mediated oxygen consumption rates were recorded to determine their activity (n = 4–8 litters/treatment, 6–8 placentas/litter). (K, L) Placental oxidative stress was determined by Western blot with anti-pH2A.X antibody, a marker of oxidative DNA damage (n = 7 litters/treatment, 1 placenta/litter). (M, N) Placental sections were stained with anti-pH2A.X antibody to determine the number of pH2A.X-positive cells in different regions of placenta (n = 5 litters/treatment, 1 placenta/litter). Two-way ANOVA with Holm–Šídák's multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001. The error bar indicates the SD. ADPR = ADP-ribose, LPS = lipopolysaccharide, NR = nicotinamide riboside.

Article Snippet: The membranes were then incubated with primary anti-ADP-ribosylation antibody (AM80-100UG; MilliporeSigma) diluted in TBS-T buffer overnight at 4°C with gentle rocking.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Colorimetric Assay, Isolation, Activity Assay, Marker, Staining

Journal: Life Science Alliance

Article Title: NAD + depletion is central to placental dysfunction in an inflammatory subclass of preeclampsia

doi: 10.26508/lsa.202302505

Figure Lengend Snippet: (A, B, C) Gene expression of inflammatory markers in human placenta biopsies from all three PE subclasses (n = 4 placentas/group). (B, C) Representative immunofluorescence images and quantification of protein ADP-ribosylation in trophoblasts of human placental tissue sections from all three PE subclasses and various control groups (n = 7–8 placentas/group). ADP-ribosylation was measured specifically within the trophoblasts highlighted using the white lines. (D, E, F, G) LC/MS quantification of human placental NAD(H) levels (n = 15–42 placentas/group). (H) Heatmap of down-regulated mitochondrial proteins in inflammatory PE3 (term) placentas compared with control term placentas (n = 5–6 per group). Shown are only proteins that met the false discovery rate threshold of 0.05. (I, J) Placental oxidative stress was determined by immunofluorescence staining of human placental tissue sections with anti-8-oxo-dG antibody and quantification by the integrated intensity/area of trophoblasts (n = 4 per group). One-way ANOVA with Holm–Šídák’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001. The error bar indicates the SD. Control = control term and preterm, CH = chronic hypertension control, PE1 = gestational parent–driven PE subclass, PE2 = hypoxia-driven PE subclass, PE3 = inflammation-driven PE subclass.

Article Snippet: Western blotting was performed as described in the Measurement of ADP-ribosylation in Cell culture measurements section using the anti-ADP-ribosylation antibody (AM80-100UG; MilliporeSigma).

Techniques: Gene Expression, Immunofluorescence, Control, Liquid Chromatography with Mass Spectroscopy, Staining

Journal: Life Science Alliance

Article Title: NAD + depletion is central to placental dysfunction in an inflammatory subclass of preeclampsia

doi: 10.26508/lsa.202302505

Figure Lengend Snippet: (A, B) Trophoblast protein ADP-ribosylation was determined by Western blot with anti-ADP-ribosylation antibody on cells treated with 10 ng/ml TNF-α and/or 150 μM–1 mM NR for 24 h (n = 3). One-way ANOVA with Holm–Šídák's multiple comparisons test. (C) Total cellular NAD + (H) and NAD + /NADH ratio quantification was performed on cells treated with or without 10 ng/ml TNF-α for 24 h (n = 3). One-tailed unpaired t test. (D, E, F, G) XFe96 Seahorse Mito stress test was performed on cells treated with 10 ng/ml TNF-α and/or 250 μM NR for 24 h. Basal, ATP-linked, and maximal mitochondrial respiration rates and extracellular acidification rates were measured (n = 4). (H, I, J, K, L) Expression of oxidative phosphorylation (OXPHOS) proteins was determined by Western blot with OXPHOS antibody cocktail on cells treated with 10 ng/ml TNF-α and/or 250 μM NR for 24 h (n = 4–7). (M, N) Trophoblast invasion capacity was determined by performing a Matrigel invasion assay on cells treated with 10 ng/ml TNF-α and/or 250 μM NR for 72 h (n = 3). Two-way ANOVA with Holm–Šídák's multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001. The error bar indicates the SD. TNF-α = tumor necrosis factor, NR = nicotinamide riboside. Source data are available for this figure.

Article Snippet: Western blotting was performed as described in the Measurement of ADP-ribosylation in Cell culture measurements section using the anti-ADP-ribosylation antibody (AM80-100UG; MilliporeSigma).

Techniques: Western Blot, One-tailed Test, Expressing, Phospho-proteomics, Invasion Assay

Journal: Life Science Alliance

Article Title: NAD + depletion is central to placental dysfunction in an inflammatory subclass of preeclampsia

doi: 10.26508/lsa.202302505

Figure Lengend Snippet: (A) Placental TNF-α levels were determined by ELISA (n = 7–9 litters/treatment, 1 placenta/litter). (B, C) Placental protein ADP-ribosylation was determined by Western blot with anti-ADP-ribosylation antibody (n = 10 litters/treatment, 1 placenta/litter). (D) Placental ADP-ribose (ADPR) levels were determined by LC/MS (n = 6–10 litters/treatment, 1 placenta/litter). (E, F, G, H) Placental NAD + (H) levels were determined by a colorimetric assay (n = 8–10 litters/treatment, 1 placenta/litter). (I, J) Placental mitochondrial function was determined by performing an oxygraph assay on isolated mitochondria. Complex I– and complex II–mediated oxygen consumption rates were recorded to determine their activity (n = 4–8 litters/treatment, 6–8 placentas/litter). (K, L) Placental oxidative stress was determined by Western blot with anti-pH2A.X antibody, a marker of oxidative DNA damage (n = 7 litters/treatment, 1 placenta/litter). (M, N) Placental sections were stained with anti-pH2A.X antibody to determine the number of pH2A.X-positive cells in different regions of placenta (n = 5 litters/treatment, 1 placenta/litter). Two-way ANOVA with Holm–Šídák's multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001. The error bar indicates the SD. ADPR = ADP-ribose, LPS = lipopolysaccharide, NR = nicotinamide riboside.

Article Snippet: Western blotting was performed as described in the Measurement of ADP-ribosylation in Cell culture measurements section using the anti-ADP-ribosylation antibody (AM80-100UG; MilliporeSigma).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Colorimetric Assay, Isolation, Activity Assay, Marker, Staining

Journal: Biological Research

Article Title: Ozone therapy mitigates parthanatos after ischemic stroke

doi: 10.1186/s40659-024-00547-5

Figure Lengend Snippet: Primary antibodies used in current study

Article Snippet: PAR , AM80 , Merck Millipore.

Techniques: