Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor

doi: 10.1073/pnas.1707965114

Figure Lengend Snippet: Expression of the enzymes regulating OCDO production and patient survival, and evidence that OCDO binds and stimulates cell proliferation through the GR and regulates GR transcriptional activity. (A–D) Kaplan–Meier representation of patient overall survival according to the indicated enzyme expression (median cut-off) using the BreastMark mining tool on 21 individual datasets (4,738 samples). Survival curves are based on Kaplan–Meier estimates and log-rank P values were calculated for differences in survival. Cox regression analysis was used to calculate HRs. (E) Kaplan–Meier representation of patient overall survival taking into account the expression of the HSD11B2, EBP (D8D7I), and DHCR7 genes using the BreastMark mining tool. (F) Representative SPR sensorgrams from three experiments showing the binding of a series of concentrations of cortisol or OCDO (µM) to the GR-LBD captured on a Biacore sensor chip: 6.25 (red); 12.5 (green); 25 (dark blue); 50 (pink); 100 (light blue). (G–J) Proliferation of the indicated tumor cells was analyzed as in Fig. 3C, n = 8. (H–J) The indicated tumor cells was treated with either the solvent vehicle (control), 5 µM OCDO, 1 µM RU486, or 5 µM OCDO plus 1 µM RU486. Data are the means (±SEM) of five separate experiments, n = 8, **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey’s posttest. (K, Upper) Cell cytosols were incubated with 10 nM [3H]-CRT and increasing concentrations of unlabeled CRT or OCDO for competition binding assays. (K, Lower) Saturation and scatchard plots analyses were performed with cell cytosols incubated with increasing concentration of [3H]-CRT in the absence or in the presence of 1 µM unlabeled CRT (nonspecific binding) or 1 µM OCDO for competitivity studies. Data are the mean (±SEM) of triplicate and are representative of three experiments. (L and M) qRT-PCR analysis of MMP1 gene expression in MDA-MB231 (L) or shC and shGR MDA-MB231 (M) cells treated either with the solvent vehicle (control), 0.5 µM cortisol, 0.1 µM DEX or 5 µM OCDO. (L and M) Data are the means ± SEM of three experiments performed in triplicate, **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey’s posttest. ns, not significant.

Article Snippet: Cox regression analysis was used to calculate HRs. ( E ) Kaplan–Meier representation of patient overall survival taking into account the expression of the HSD11B2, EBP (D8D7I), and DHCR7 genes using the BreastMark mining tool. ( F ) Representative SPR sensorgrams from three experiments showing the binding of a series of concentrations of cortisol or OCDO (μM) to the GR-LBD captured on a Biacore sensor chip: 6.25 (red); 12.5 (green); 25 (dark blue); 50 (pink); 100 (light blue). ( G – J ) Proliferation of the indicated tumor cells was analyzed as in , n = 8. ( H – J ) The indicated tumor cells was treated with either the solvent vehicle (control), 5 μM OCDO, 1 μM RU486, or 5 μM OCDO plus 1 μM RU486.

Techniques: Expressing, Activity Assay, Binding Assay, Solvent, Control, Incubation, Concentration Assay, Quantitative RT-PCR, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor

doi: 10.1073/pnas.1707965114

Figure Lengend Snippet: Binding of the indicated ligands to ERα and LXRs, extinction of GR and LXRβ expression in the indicated BC cells and evidence that OCDO does not stimulate cell proliferation through the LXRβ. (A–F) Representative SPR sensorgrams from three experiments showing the binding of a series of concentrations of OCDO, 22(R)HC, or 17β-estradiol to the indicated receptor-LBD, captured on a Biacore sensor chip. Concentrations: 6.25 µM (red), 12.5 µM (green), 25 µM (dark blue), 50 µM (pink), 100 µM (light blue). Each sensorgram (expressed in RUs as a function of time in seconds) represents a differential response where the response on an empty reference channel (Fc1) was subtracted. The affinity constant (KD) is determined at equilibrium by the BIAevaluation software. (G and H) GR expression (G) or LXRβ expression (H) in MCF7 or MDA-MB-231 cells was knocked down using either shRNA against the GR (clones shGR5 and shGR6), or the LXRβ (clones shLXR3 and LXR4), or control shRNA (shC) and the expression of receptors was analyzed by immunoblotting. The blots are representative of three experiments. (I) Effect of solvent vehicle (control) or OCDO (5 or 10 µM) on the proliferation of the indicated tumor cells using a colorimetric BrDU immunoassay. (J) Effects of solvent vehicle (control) or OCDO (5 µM) or DEX 0.1 µM, or OCDO (5 µM) + DEX 0.1 µM on the proliferation of the indicated tumor cells using a colorimetric BrDU immunoassay. (I and J) Data are the means (±SEM) of five separate experiments, (n = 8), **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey’s posttest. ns, not significant.

Article Snippet: Cox regression analysis was used to calculate HRs. ( E ) Kaplan–Meier representation of patient overall survival taking into account the expression of the HSD11B2, EBP (D8D7I), and DHCR7 genes using the BreastMark mining tool. ( F ) Representative SPR sensorgrams from three experiments showing the binding of a series of concentrations of cortisol or OCDO (μM) to the GR-LBD captured on a Biacore sensor chip: 6.25 (red); 12.5 (green); 25 (dark blue); 50 (pink); 100 (light blue). ( G – J ) Proliferation of the indicated tumor cells was analyzed as in , n = 8. ( H – J ) The indicated tumor cells was treated with either the solvent vehicle (control), 5 μM OCDO, 1 μM RU486, or 5 μM OCDO plus 1 μM RU486.

Techniques: Binding Assay, Expressing, Software, shRNA, Clone Assay, Control, Western Blot, Solvent

Journal: Signal Transduction and Targeted Therapy

Article Title: A deep learning-driven discovery of berberine derivatives as novel antibacterial against multidrug-resistant Helicobacter pylori

doi: 10.1038/s41392-024-01895-0

Figure Lengend Snippet: Mechanism of action and direct targets exploration on compound 8 . a , b Images for morphology of H. pylori under electron microscope ( a ) SEM images of H. pylori treated without (upper) or with (lower) 8 . b TEM images of H. pylori treated without (upper) or with (lower) 8 . c The structure of the active photoaffinity probe 8 - O . d Cy3-labeled target proteins were identified using fluorescent gel imaging. SecA ( e ) and BamD ( f ) were pulled down from H. pylori by using probe 8 - O in immunoblot assay. SecA and BamD pulled down by 8 - O were competitively inhibited by 8 . The recombinant SecA ( g ) and BamD ( h ) proteins pulled down by 8 - O were competitively inhibited by 8 . Surface plasmon resonance (SPR) sensorgrams obtained on SecA ( i )/BamD ( j )-coated chips at different concentrations of 8 . The thermal stability of SecA ( k )/BamD ( l ) proteins with or without 8 -treatment ( n = 3)

Article Snippet: Surface plasmon resonance (SPR) sensorgrams obtained on SecA ( i )/BamD ( j )-coated chips at different concentrations of 8 .

Techniques: Microscopy, Labeling, Imaging, Western Blot, Recombinant, SPR Assay