Journal: mSphere

Article Title: An alteration in the expression of cell wall structural proteins increases cell surface exposure of adhesins to promote virulence in Candida glabrata

doi: 10.1128/msphere.00910-24

Figure Lengend Snippet: The reduced cell surface exposure of Epa1 in the tir2-5 awp6-7 sextuple mutant. ( A ) Fluorescence-activated cell sorting (FACS) analysis of HA-Epa1 fusion proteins on the cell surface in WT strain (black) and tir2-5 awp6-7 sextuple mutant (red). C. glabrata cells carrying HA-Epa1 from the TEF1 promoter were incubated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% serum at 37°C in 5% CO 2 . Surface exposure was monitored by immunofluorescence after labeling with anti-HA antibody and fluoresceine isothiocyanate (FITC)-conjugated secondary goat anti-mouse antibody. No primary antibody control was included. Average fluorescence of the population was indicated. Plots are representative of data collected in two independent replicate experiments. ( B ) Western analysis of HA-Epa1 fusion protein in the cell wall fraction of WT and tir2-5 awp6-7 sextuple mutant cells incubated in DMEM medium with 10% serum at 37°C in 5% CO 2 . Band intensities were quantified and shown as the ratio of indicated protein vs tubulin. Representative plots of three independent experiments are shown. ( C ) Real-time quantitative reverse transcription PCR (qRT-PCR) analysis for EPA1 expression in C. glabrata cultured as described in ( B ). The expression levels of EPA1 were showed as means ± SD. n = 3 biologically independent experiments. ( D ) Adhesion assay on epithelial cell monolayers. C. glabrata cells of wild type and indicated mutant strains were inoculated with A549 cells in DMEM medium supplemented with 10% serum. After incubation for 4 h, non-adherent Candida cells were removed by washing with phosphate-buffered saline (PBS). The numbers of adherent Candida cells are represented as means ± SD from three independent experiments. ( E ) Cytotoxicity assays. A549 cell damage following treatment with wild type, tir2-5 quadruple mutant, awp6-7 double mutant, or tir2-5 awp6-7 sextuple mutant was determined after co-incubation for 24 h. Relative LDH activity (%) against A549 cells without C. glabrata cells was calculated. Mean data ± SD from three independent experiments was plotted. ( C–E ) Significance was determined using the two-tailed unpaired Student’s t -test. ns, nonsignificant. ** P < 0.01, *** P < 0.001.

Article Snippet: Cell wall proteins were then released from the washed cell wall material by treatment with Endo-1,3-β-glucanase (TargetMoI) and probed for HA-Epa1 using anti-HA antibody (MBL).

Techniques: Mutagenesis, Fluorescence, FACS, Incubation, Modification, Immunofluorescence, Labeling, Control, Western Blot, Reverse Transcription, Quantitative RT-PCR, Expressing, Cell Culture, Cell Adhesion Assay, Saline, Activity Assay, Two Tailed Test

Journal: Translational Neuroscience

Article Title: Behavior and Hippocampal Epac Signaling to Nicotine CPP in Mice

doi: 10.1515/tnsci-2019-0041

Figure Lengend Snippet: Hippocampal α7 nAChR, PKA, Epac, Rap1, and pCREB in mice after nicotine place conditioning There was no difference in hippocampal a7 nAChR in male (A) and female mice (B). There was no difference in PKA in male (C) and female mice (D). There was no difference in Epac1 in male (E) and female mice (F). Epac2 level was significantly increased in male (G) and female (H) mice. Rap1 expression was increase in female mice (J), but not in male mice (I). Consistently, p-CREB/CREB ratio expression was decreased in female mice (L), but not in male mice (K). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, n = 4 in each group.

Article Snippet: Primary antibodies of Epac1 (4155S, 1:500), Epac2 (4156S, 1:1000), Rap1A/1B (4938S, 1:1000), CREB (9197S, 1:1000), and phospho-CREB (9198S, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing

Journal: Pharmacological research

Article Title: Ginsenoside Ro prevents endothelial injury via promoting Epac1/AMPK- mediated mitochondria protection in early diabetic retinopathy.

doi: 10.1016/j.phrs.2024.107562

Figure Lengend Snippet: Fig. 6. Ro protected hRMECs from AGEs-induced injury via the EPAC1/AMPK signaling pathway. A. Venn diagram drawn by the intersection of the genes that increased in the control group compared to the AGEs group and the genes that increased in the Ro treatment group compared to the AGEs group. B. Expression levels of Epac1 in control, model, and Ro-treated groups. C. The top 10 of KEGG passway Signaling Pathways. D, E. Representative immunoblots showed Epac1 and p- AMPK protein level in hRMECs. F, G. Images of Epac1 staining in hRMECs. The fluorescence intensity was quantified by Image J. Scale bar, 5um. The results are expressed as mean ± SD, #P < 0.05 and ##P < 0.01 vs. the control, **P < 0.001 vs. AGEs group.

Article Snippet: Then, the membrane was blocked using 5 % skim milk solution in TBST for 2 h at room temperature and incubated with corresponding primary antibodies overnight at 4°C: β-actin (ABclonal, AC038, 1:2000); EPAC1 (Proteintech, 12572–1- AP, 1:2000); p-AMPK (Abcam, AB133448, 1:2000); AMPK (Abcam, AB32047, 1:2000); Bcl-2 (ABclonal, A0208, 1:2000); CD31/PECAM1 (ABclonal, A0378, 1:1000); p-DRP-1 (ABclonal, AP1353, 1:1000); DRP1 (ABclonal, A21968, 1:1000); PINK1 (ABclonal, A25301, 1:2000); Parkin (ABclonal, A0968, 1:1000)LC3 (Sigma-Aldrich, L8918, 1:2000); SQSTM1/p62 (ABclonal, 1:2000); ATG5 (ABclonal, A0203, 1:1000); ATG12 (ABclonal, A22788, 1:1000); Beclin-1 (ABclonal, A7353, 1:2000).

Techniques: Control, Expressing, Protein-Protein interactions, Western Blot, Staining, Fluorescence

Journal: Pharmacological research

Article Title: Ginsenoside Ro prevents endothelial injury via promoting Epac1/AMPK- mediated mitochondria protection in early diabetic retinopathy.

doi: 10.1016/j.phrs.2024.107562

Figure Lengend Snippet: Fig. 8. Ro regulates Epac1/AMPK signaling pathway to ameliorate DR in diabetic mice. A. The mitochondria in mouse retinal microvascular endothelial cells were analysed by TEM. Red arrows indicate mitochondria. Scale bar, 1μm. B, C. Expression levels of Epac1 protein in mouse retinal cells, and DAPI marker nuclei. Representative fluorescence images of Epac1 were captured by TissueFAXS Cytometry. Scale bars: 200 µm. D, E. Expression levels of p-AMPK protein in mouse retinal cells, and DAPI marker nuclei. Representative fluorescence images of p-AMPK were captured by TissueFAXS Cytometry. Scale bars: 200 µm. The results are expressed as mean ± SD, #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. the control, *P < 0.05, **P < 0.001 and ***P < 0.001 vs. AGEs group.

Article Snippet: Then, the membrane was blocked using 5 % skim milk solution in TBST for 2 h at room temperature and incubated with corresponding primary antibodies overnight at 4°C: β-actin (ABclonal, AC038, 1:2000); EPAC1 (Proteintech, 12572–1- AP, 1:2000); p-AMPK (Abcam, AB133448, 1:2000); AMPK (Abcam, AB32047, 1:2000); Bcl-2 (ABclonal, A0208, 1:2000); CD31/PECAM1 (ABclonal, A0378, 1:1000); p-DRP-1 (ABclonal, AP1353, 1:1000); DRP1 (ABclonal, A21968, 1:1000); PINK1 (ABclonal, A25301, 1:2000); Parkin (ABclonal, A0968, 1:1000)LC3 (Sigma-Aldrich, L8918, 1:2000); SQSTM1/p62 (ABclonal, 1:2000); ATG5 (ABclonal, A0203, 1:1000); ATG12 (ABclonal, A22788, 1:1000); Beclin-1 (ABclonal, A7353, 1:2000).

Techniques: Expressing, Marker, Fluorescence, Cytometry, Control

Journal: Pharmacological research

Article Title: Ginsenoside Ro prevents endothelial injury via promoting Epac1/AMPK- mediated mitochondria protection in early diabetic retinopathy.

doi: 10.1016/j.phrs.2024.107562

Figure Lengend Snippet: Fig. 9. Retina-specific deletion of Epac1 reverses the therapeutic effects of Ro. A, E. Retinal thickness was determined by OCT. Scale bar, 100um. B, F. Detection of changes in retinal blood vessels in mice by FFA. C, G. Representative images of H&E staining of retinal tissue from the mice. Scale bar, 50μm. D. The mitochondria in mouse retinal microvascular endothelial cells were analysed by TEM. Red arrows indicate mitochondria. Scale bar, 1μm. H, I. Expression levels of p- AMPK protein in mouse retinal cells, and DAPI marker nuclei. Representative fluorescence images of p-AMPK were captured by TissueFAXS Cytometry. Scale bars: 200 µm. The results are expressed as mean ± SD, #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. the control, *P < 0.05, **P < 0.001 and ***P < 0.001 vs. AGEs group.

Article Snippet: Then, the membrane was blocked using 5 % skim milk solution in TBST for 2 h at room temperature and incubated with corresponding primary antibodies overnight at 4°C: β-actin (ABclonal, AC038, 1:2000); EPAC1 (Proteintech, 12572–1- AP, 1:2000); p-AMPK (Abcam, AB133448, 1:2000); AMPK (Abcam, AB32047, 1:2000); Bcl-2 (ABclonal, A0208, 1:2000); CD31/PECAM1 (ABclonal, A0378, 1:1000); p-DRP-1 (ABclonal, AP1353, 1:1000); DRP1 (ABclonal, A21968, 1:1000); PINK1 (ABclonal, A25301, 1:2000); Parkin (ABclonal, A0968, 1:1000)LC3 (Sigma-Aldrich, L8918, 1:2000); SQSTM1/p62 (ABclonal, 1:2000); ATG5 (ABclonal, A0203, 1:1000); ATG12 (ABclonal, A22788, 1:1000); Beclin-1 (ABclonal, A7353, 1:2000).

Techniques: Staining, Expressing, Marker, Fluorescence, Cytometry, Control

Journal: Journal of ethnopharmacology

Article Title: Anemarrhena asphodeloides Bunge and Phellodendri Chinensis Cortex inhibits the PTGS2/EP2/cAMP/Epac1 signaling pathway to reduce microglial M1 polarization, thereby blocking chronic stress-induced depression-like behavior.

doi: 10.1016/j.jep.2025.119792

Figure Lengend Snippet: Fig. 8. Neomangiferin alleviates the release of proinflammatory factors in BV2 cells by inhibiting PTGS2/EP2/cAMP/Epac1 signals. (A) representative immunofluorescence staining of EP2 and Epac1, and (B) fluorescence area (n = 3). (C–D) Protein expression changes of EP2 and Epac1 (n = 3). The contents of PGE2 and cAMP in BV2 cells were determined by E-F Elisa kit (n = 3). (G) Representative images of zebrafish embryos in Neomangiferin experiment (n = 10). Data are expressed as mean±SD. *** means P < 0.001,** means P < 0.01,* means P < 0.05, ns means P > 0.05.

Article Snippet: Seal with 5 % skim milk powder at room temperature for 2 h. Primary antibody β-actin (rabbit, 1:5000; bs-00R,Bioss, China), GAPDH(rabbit, 1:10,000; 10494- 1-AP,Proteintech, China), HSP90(rabbit, 1:5000; 13171-1-AP,Proteintech, China), Arg-1 (rabbit, 1:1000; 16001-AP, Proteintech, China), iNOS (rabbit, 1:1000; ab283655, Abcam, UK), PTGS2 (mouse, 1:400; 66351-1-Ig, Proteintech, China), EP2 (rabbit, 1:1000; HA721380, HUABIO, China), EPAC1 (rabbit, 1:1000; A02483-3, Boster, China), incubated overnight at 4 ◦C.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay