Journal: Nature Communications

Article Title: Translocation of vaginal microbiota is involved in impairment and protection of uterine health

doi: 10.1038/s41467-021-24516-8

Figure Lengend Snippet: a Study design of one-to-one exchange between the vaginal (V) microbiota of rats. Two groups of female Brown Norway (BN, n = 12) and Sprague Dawley (SD, n = 12) rats were used as donors and recipients, and the uterine (U) lavage fluid of each rat was sampled 3 weeks after exchange and was used for 16S rRNA gene sequencing. b Chao 1 index of the uterine microbiome of each rat before (Pre-) and after (Post-) exchanging the vaginal microbiota. c Shannon diversity index of the uterine microbiome in the pre- and post-exchange rats. The violin with box plot shows the median and interquartile range, and the width of the violin represents the density distribution. P values were determined by two-tailed Wilcoxon test. d Principal coordinate analysis (PCoA) of the uterine samples of the rats before and after vaginal microbiota exchange. The significance of separation of two clusters was measured with the Adonis test ( P < 0.05). e Variations in the diversity and structure of the uterine microbiome in the rats before and after exchange. The taxonomic classification and profiling are shown at the phylum level, with each wave represents a family. Color bar represents the relative abundance of each family per 10 5 reads. f Study design of vaginal microbiota transplantation (VMT) from women to rats. Vaginal lavage fluids were collected from 10 women each in the healthy, chronic endometritis and bacterial vaginosis groups and were transplanted into the vagina of SD rats ( n = 10) after 1 week of antibiotic treatment. g The mRNA expression and cytokine levels of inflammatory factors in the endometrial tissues of VMT rats. For each group, n = 10. The upper, center, and lower lines among points show the means ± s.e.m in mRNA expression. The width of the violin represents the density distribution of cytokine levels, and the upper, center and lower lines among points represent upper quartile, median and lower quartile, respectively. P values were determined by two-tailed Student’s t -test. h The edematous uterine body and symptoms of the endometrial tissues in the VMT rats. The 25× and 50× fields of view show the areas within the solid and dashed frames of the 5× field of view. The black arrows under 5×, 25×, and 50× fields of view show hyperplastic glands, endometrial epithelium, and inflammatory granulocytes in the uterus of rats transplanted with the vaginal microbiota of women with endometritis, respectively. i Immunofluorescence assay illustrating the TNF-α and CD38 signals in the endometrial tissue of the VMT rats. The white arrow shows signals from inflammatory factors. For h , i images are representative of three independent experiments with similar results.

Article Snippet: Immunofluorescence (IF) was performed using anti-TNF-α (ab109322, Abcam, Cambridge, MA) and anti-CD38 (bs-0979R, Bioss Antibodies, Beijing, China) antibodies.

Techniques: Sequencing, Two Tailed Test, Transplantation Assay, Expressing, Immunofluorescence

Journal: Nature Communications

Article Title: Translocation of vaginal microbiota is involved in impairment and protection of uterine health

doi: 10.1038/s41467-021-24516-8

Figure Lengend Snippet: a Study design of the vaginal microbiota transplantation (VMT) challenge experiment involving injection of Prevotella bivia ( P. bivia , P.b .) or Clostridium perfringens ( C. perfringens , C.p .) into the vagina of SD rats. The rats in the blank control (BC) group were injected with PBS instead of bacteria. b The mRNA expression of inflammatory factors in the endometrial tissue and the diameter of the uterine body in the P.b . ( n = 17), C.p . ( n = 16) and BC ( n = 10) rats. P values were determined by two-tailed Student’s t -test, and the lines among points show the means. c The correlation between mRNA expression of inflammatory factors and uterine diameter. The shadow around the linear regression trendline shows the 95% confidence interval (CI). d The uterine bodies and hematoxylin-eosin staining of the endometrial tissues in the C.p . and BC rats. The 25× and 50× fields of view show the areas within the solid and dashed frames of the 5× field of view. The blue arrow under a 5× field of view shows bleeding with blood cells and inflammatory cells (including lymphocyte cells and inflammatory granulocytes) in the uterine cavity. The black arrows under a 25× field of view show hyperplastic endometrial epithelium. The red and yellow arrows under a 50× field of view refer to plasma cells and inflammatory granulocytes, respectively. e Immunofluorescence assay illustrating the TNF-α and CD38 signals in the endometrial tissue of the C.p . and BC rats. f Fluorescence in situ hybridization (FISH) of total bacteria (green) and Clostridium (red) in the uterine cavity of the C.p . rats. g Study design of injecting C.p ., Lactobacillus murinus ( L. murinus , L.m .), supernatant of C. perfringens (SC) and PBS (BC) into the vagina of SD rats. h The mRNA expression of inflammatory factors in endometrial tissue, the biomass of vaginal and uterine Clostridium and their correlations with uterine diameter in VMT rats. i Principal coordinate analysis (PCoA) of the uterine microbiome of the C.p ., L.m ., SC, and BC rat groups. j Relative abundance of Clostridium , Lactobacillus , Prevotella , and Bacteroides in the uterine microbiota of the C.p . ( n = 15), L.m . ( n = 15), SC ( n = 15) and BC ( n = 10) rat groups. Data are presented as means ± s.e.m. For d – f images are representative of three independent experiments with similar results. For c , h linear regression was used to analysis the correlation between two pairs of data, the shadow around the trendline shows the 95% confidence interval (CI). For h , j P values were determined by one-way ANOVA with Tukey’s multiple comparison post-hoc test, and data are presented as means ± s.e.m, and ns represents P < 0.05.

Article Snippet: Immunofluorescence (IF) was performed using anti-TNF-α (ab109322, Abcam, Cambridge, MA) and anti-CD38 (bs-0979R, Bioss Antibodies, Beijing, China) antibodies.

Techniques: Transplantation Assay, Injection, Expressing, Two Tailed Test, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Western blot analysis confirmed the overexpression of CD38 in A549-CD38 cells compared with A549-Plvx cells. B Relative ROS levels in A549-Plvx and A549-CD38 cells ( n = 3). C and D NAD + and NADP + levels in A549-Plvx and A549-CD38 cells were determined by peak areas from the metabolomics analysis ( n = 3). E Relative ROS levels of A549 cells treated with or without 100 nM FK866 for 12 h ( n = 3). F A549 cells were treated with or without 100 nM FK866 for 12 h. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). G The relative transcription levels of DHFR in untreated and 100 nM FK866-treated A549 cells ( n = 3). H The relative ROS levels in A549-CD38 cells treated with or without 1 mM NMN for 12 h ( n = 3). I A549-Plvx and A549-CD38 cells were treated with or without 1 mM NMN. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). J The relative transcription levels of DHFR in A549-Plvx and A549-CD38 cells treated with or without 1 mM NMN ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Western Blot, Over Expression, Control

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Relative ROS levels were measured in A549 cells treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h ( n = 3). B A549 cells were treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). C The transcription levels of DHFR in A549 cells treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h ( n = 3). D Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells pre-treated with 6 mM NAc for 6 h or not. Graphs represent the quantification of the blots ( n = 3). E Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells co-treated with 100 nM Baf-A1 or not. Graphs represent the quantification of the blots ( n = 3). F Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells pre-treated with 10 μM MG132 for 4 h or not. Graphs represent the quantification of the blots ( n = 3). G Western blot images of DHFR and Actin (loading control) in DMSO, NAc (6 mM, 6 h), Baf-A1 (100 nM, 6 h), and MG132 (10 μM, 4 h)-treated A549-CD38 cells. Graphs represent the quantification of the blots ( n = 3). Data were shown as mean ± SD and analyzed by one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Control, Western Blot

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A and B The survival rate of A549-Plvx and A549-CD38 cells treated with different concentrations of Erastin ( A ) or RSL3 ( B ) ( n = 3). C and D The survival rate of A549-Plvx and A549-CD38 cells treated with Erastin (5, 2.5 μM) or RSL3 (2.5, 1.25 μM) with or without 1 mM NMN co-treatment for 12 h ( n = 3). E and F The survival rate of A549 cells under Erastin or RSL3 treatment co-treated with or without 100 nM methotrexate (MTX) ( n = 3). G and H The survival rate of A549-CD38 cells under Erastin or RSL3 treatment before or after replenishing DHFR ( n = 3). I The survival rate of A549-CD38 cells treated with RSL3 (0.625 μM, 12 h), RSL3 (0.625 μM) + Baf-A1 (100 nM, co-treatment with RSL3 for 12 h), and RSL3 (0.625 μM) + MG132 (10 μM, pre-treatment for 4 h) ( n = 3). J The survival rate of A549-CD38 cells treated with DMSO (Control), Baf-A1 (100 nM), and MG132 (10 μM, pre-treatment for 4 h) for 12 h ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Control

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Western blot analysis of CD38, DHFR, and Actin (loading control) in BMDMs from young and aged mice, respectively. Graphs represent the quantification of the blots ( n = 3). B The relative ROS levels of BMDMs from young and aged mice ( n = 3). C and D The mRNA expression levels of cd38 and dhfr in BMDMs from young and aged mice, respectively ( n = 3). E and F The survival rate of BMDMs from young and aged mice under Erastin ( E ) or RSL3 ( F ) treatment ( n = 3). G The survival rate of BMDMs from young and aged mice under Erastin (20 μM), Erastin (20 μM) + NMN (1 mM), Erastin (20 μM) + DFO (50 μM), Erastin (20 μM) + Fer-1 (1 μM) treatment for 12 h ( n = 3). H The survival rate of BMDMs from young and aged mice under RSL3 (0.625 μM) or + RSL3 (0.625 μM) + NMN (1 mM), RSL3 (0.625 μM) + DFO (50 μM), RSL3 (0.625 μM) + Fer-1 (1 μM) treatment for 12 h ( n = 3). I The survival rate of BMDMs from aged mice under RSL3 (0.625 μM, 12 h), RSL3 (0.625 μM) + Baf-A1 (100 nM, co-treatment with RSL3 for 12 h), and RSL3 (0.625 μM) + MG132 (10 μM, pre-treatment for 4 h) ( n = 3). J The survival rate of BMDMs from aged mice under DMSO (Control), Baf-A1 (100 nM), and MG132 (10 μM, pre-treatment for 4 h) treatment for 12 h ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Western Blot, Control, Expressing

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Western blot analysis confirmed the overexpression of CD38 in A549-CD38 cells compared with A549-Plvx cells. B Relative ROS levels in A549-Plvx and A549-CD38 cells ( n = 3). C and D NAD + and NADP + levels in A549-Plvx and A549-CD38 cells were determined by peak areas from the metabolomics analysis ( n = 3). E Relative ROS levels of A549 cells treated with or without 100 nM FK866 for 12 h ( n = 3). F A549 cells were treated with or without 100 nM FK866 for 12 h. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). G The relative transcription levels of DHFR in untreated and 100 nM FK866-treated A549 cells ( n = 3). H The relative ROS levels in A549-CD38 cells treated with or without 1 mM NMN for 12 h ( n = 3). I A549-Plvx and A549-CD38 cells were treated with or without 1 mM NMN. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). J The relative transcription levels of DHFR in A549-Plvx and A549-CD38 cells treated with or without 1 mM NMN ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Western Blot, Over Expression, Control

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Relative ROS levels were measured in A549 cells treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h ( n = 3). B A549 cells were treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). C The transcription levels of DHFR in A549 cells treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h ( n = 3). D Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells pre-treated with 6 mM NAc for 6 h or not. Graphs represent the quantification of the blots ( n = 3). E Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells co-treated with 100 nM Baf-A1 or not. Graphs represent the quantification of the blots ( n = 3). F Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells pre-treated with 10 μM MG132 for 4 h or not. Graphs represent the quantification of the blots ( n = 3). G Western blot images of DHFR and Actin (loading control) in DMSO, NAc (6 mM, 6 h), Baf-A1 (100 nM, 6 h), and MG132 (10 μM, 4 h)-treated A549-CD38 cells. Graphs represent the quantification of the blots ( n = 3). Data were shown as mean ± SD and analyzed by one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Control, Western Blot

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A and B The survival rate of A549-Plvx and A549-CD38 cells treated with different concentrations of Erastin ( A ) or RSL3 ( B ) ( n = 3). C and D The survival rate of A549-Plvx and A549-CD38 cells treated with Erastin (5, 2.5 μM) or RSL3 (2.5, 1.25 μM) with or without 1 mM NMN co-treatment for 12 h ( n = 3). E and F The survival rate of A549 cells under Erastin or RSL3 treatment co-treated with or without 100 nM methotrexate (MTX) ( n = 3). G and H The survival rate of A549-CD38 cells under Erastin or RSL3 treatment before or after replenishing DHFR ( n = 3). I The survival rate of A549-CD38 cells treated with RSL3 (0.625 μM, 12 h), RSL3 (0.625 μM) + Baf-A1 (100 nM, co-treatment with RSL3 for 12 h), and RSL3 (0.625 μM) + MG132 (10 μM, pre-treatment for 4 h) ( n = 3). J The survival rate of A549-CD38 cells treated with DMSO (Control), Baf-A1 (100 nM), and MG132 (10 μM, pre-treatment for 4 h) for 12 h ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Control

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Western blot analysis of CD38, DHFR, and Actin (loading control) in BMDMs from young and aged mice, respectively. Graphs represent the quantification of the blots ( n = 3). B The relative ROS levels of BMDMs from young and aged mice ( n = 3). C and D The mRNA expression levels of cd38 and dhfr in BMDMs from young and aged mice, respectively ( n = 3). E and F The survival rate of BMDMs from young and aged mice under Erastin ( E ) or RSL3 ( F ) treatment ( n = 3). G The survival rate of BMDMs from young and aged mice under Erastin (20 μM), Erastin (20 μM) + NMN (1 mM), Erastin (20 μM) + DFO (50 μM), Erastin (20 μM) + Fer-1 (1 μM) treatment for 12 h ( n = 3). H The survival rate of BMDMs from young and aged mice under RSL3 (0.625 μM) or + RSL3 (0.625 μM) + NMN (1 mM), RSL3 (0.625 μM) + DFO (50 μM), RSL3 (0.625 μM) + Fer-1 (1 μM) treatment for 12 h ( n = 3). I The survival rate of BMDMs from aged mice under RSL3 (0.625 μM, 12 h), RSL3 (0.625 μM) + Baf-A1 (100 nM, co-treatment with RSL3 for 12 h), and RSL3 (0.625 μM) + MG132 (10 μM, pre-treatment for 4 h) ( n = 3). J The survival rate of BMDMs from aged mice under DMSO (Control), Baf-A1 (100 nM), and MG132 (10 μM, pre-treatment for 4 h) treatment for 12 h ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Western Blot, Control, Expressing

Journal: Pharmacological research

Article Title: Targeting CD38/cyclic ADP-ribosyl cyclase as a novel therapeutic strategy for identification of three potent agonists for leukopenia treatment.

doi: 10.1016/j.phrs.2024.107068

Figure Lengend Snippet: Fig. 1. Model construction of virtual screening and discovery of candidate compounds. (A) Prediction score of 41 compounds of Sanguisorba officinalis L. binding with the ADP ribosyl cyclase structure of CD38. (B) Molecular docking simulation reveals the interaction between compounds (ZGSII, DMA, CG, PB3, OA, BA) and the ADP ribosyl cyclase structure of CD38. (C-H) The RMSD and RMSF values were obtained for the ADP ribosyl cyclase structure complexed with ZGSII, DMA, CG, PB3, OA, and BA, respectively.

Article Snippet: Primary antibodies were as follows: CD38 (Proteintech, IL, USA, 60006–1-lg), GAPDH (Proteintech, USA, 60004–1-lg), interferon-gamma (IFN-γ) (Proteintech, USA, 15365–1-AP), NFAT2 (Proteintech, USA, 66963–1-lg), β-actin (CST, MA, USA, 3700 S), and histone H3 (Proteintech, USA, 17168–1-AP).

Techniques: Binding Assay