Journal: Cells

Article Title: The Drosophila RNA Helicase Belle (DDX3) Non-Autonomously Suppresses Germline Tumorigenesis Via Regulation of a Specific mRNA Set

doi: 10.3390/cells9030550

Figure Lengend Snippet: Comparative analysis of candidate targets of Belle CLIP-seq with the results of published screens. ( A ) A Venn diagram shows the overlap of genes, RNAi knockdowns of which provide the morphological defects similar to RNAi belKD in the testes according to data of three independent screens [ , , ], and which were determined in our CLIP-seq analysis as putative mRNA targets of Belle. Knockdowns of four genes, not1, rpn7, rho1, and caf1-55 , provide the same morphological defects in the testes, according to data from all three independent screens. ( B ) Western blot analysis of testis lysates of c587-GAL4>UAS-bel RNAi males, nos-GAL4>UAS-bel RNAi males, and the corresponding driver lines as controls. Anti-actin antibodies were used as a loading control. The values (obtained by using ImageJ software) were normalized to the loading control level (actin) and then to the values obtained for the control lysates. Mean values and standard errors are presented for at least three independent experiments. We revealed a decrease in the level of Not1 and Caf1-55 proteins in the testes with belKDs ; however, we did not detect significant changes in the level of Rho1. ( C – E ) Immunostaining of fixed testis preparations of males with RNAi knockdown of not1 using soma-specific driver tj-GAL4 . Testes of newly eclosed males were stained with anti-Vasa (red) and anti-Tj (violet) antibodies; chromatin was stained by DAPI (4′,6-diamidino-2-phenylindole, blue). Scale bars are 30 µm. ( C ) Control testes contained a wild-type array of cells at all stages of spermatogenesis. ( D ) Most of not1KD testes are filled by tumor-like clusters of small germ cells and somatic cyst cells placed separately. ( E ) In the remaining not1KD testes clusters of small germ cells were found along with differentiating germ cells. ( F ) Interaction cluster analysis of genes from the full list of Belle CLIP-targets shared with the results of the screen studies was performed by STRING database tools. Only 17 of 22 genes form five interacting clusters with high confidence settings (interaction score 0.7). The thickness of the line between genes (color balls) reflects the relative probability of interaction. See for a detailed description.

Article Snippet: For Western blot analysis, the following antibodies were used: murine monoclonal anti-β-actin antibody ab8224 (Abcam), 1:4000; rabbit polyclonal anti-Piwi 2464 antibodies [ ], 1:2000; goat polyclonal anti-STAT dN-17 antibodies (Santa Cruz Biotechnology), 1:100; goat polyclonal anti-Hedgehog dD-12 antibodies (Santa Cruz Biotechnology); 1:100; rat monoclonal anti-Cubitus interruptus (Ci) 2A1 antibody (DSHB), 1:100; murine monoclonal anti-Rac1 antibody 610,651 (BD Transduction Laboratories), 1:1000; murine monoclonal anti-Gbb antibody 3D-6 (DSHB), 1:50; rabbit monoclonal anti-Dpp antibody (Santa-Cruz Biotechnology), 1:100; rabbit monoclonal anti-pERK antibody (Cell Signaling), 1:2000; rabbit monoclonal anti-pSmad antibody ab52903 (Abcam), 1:500; murine monoclonal anti-Not1 2G5 antibody [ ], 1:250; rabbit polyclonal anti-Caf1-55 antibodies ab1766 (Abcam), 1:2000; and murine monoclonal anti-Rho1 antibody p1D9 (DSHB), 1:50.

Techniques: Western Blot, Software, Immunostaining, Staining

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: The differential activation of CD14 and CD16 monocytes responding to endothelial and epithelial dysfunction. The profile scores of molecules mediating the interplays of classic monocytes with vascular endothelial cells (A-B) and non-classic monocytes with alveolar epithelial cells (C—D). E. Immunostaining for CDH5 and γH2AX in coronary artery cryo-section between KD patient and healthy donor, indicating endothelial injuries in KD. F. Expression level of CCR1 , DYSF , SELL , LMNB1, and XAF1 in CD14 classical monocytes among five groups. G. UMAP projection of CCR1 , DYSF , SELL , LMNB1, and XAF1 positive cells, respectively . H. UMAP projection of CCR1 , SELL and XAF1 triple positive monocytes among five groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Wilcoxon rank-sum test, adjusted for Bonferroni post hoc test, had been applied.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Activation Assay, Immunostaining, Expressing

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: SELL+/CCR1+/XAF1+ CD14 monocytes enhanced the adhesion and damages to endothelial cells. A. Flow cytometry identified the higher percentages and median fluorescence intensity of SELL, CCR1, and LMNB1 in KD and COV. B. Immunostaining for CD14 and DYSF, XAF1 in isolated PBMCs. C. The ratio of DYSF positive CD14 monocytes in total classic monocytes among KD, COV, FLU, and healthy donors. And the median fluorescence intensity of XAF1 in CD14 monocytes among KD, FLU, and healthy donors. D-E. THP-1 had been stained with Calcein AM and transfected siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 before co-culture with HUVECs. Then the numbers of adhesion THP-1 with HUVECs were counted by every 20× field view. F. The expressions of TNFa and IL6 in HUVECs, and the ratio of γH2AX + cells in HUVECs after co-cultured with THP-1, which had been transfected with siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 . G. The inhibition of SELL , CCR1 and XAF1 together in THP-1 significantly reduced the adhesion between monocytes and endothelial cells. H. Collaborated inhibiton of SELL , CCR1 and XAF1 in THP-1 decreased the expression of IL6 and TNF-α in HUVECs with lower ratio of rH2AX+ cells after co-culture with THP-1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way analysis of variance with Bonferroni post hoc test was performed to analyze data. Bar, 100 μm.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Flow Cytometry, Fluorescence, Immunostaining, Isolation, Staining, Transfection, Co-Culture Assay, Cell Culture, Inhibition, Expressing

Journal: Nature Communications

Article Title: Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis

doi: 10.1038/s41467-017-00834-8

Figure Lengend Snippet: Anti-inflammatory effects of CLT and CLT-AN in anti-Thy1.1 nephritic rats. a – d Real-time PCR analysis of renal mRNA levels of MCP-1 ( a ), ICAM-1 ( b ), IL-6 ( c ), IL-1β ( d ) in anti-Thy1.1 nephritic rats on day 1 after treatment with CLT or CLT-AN. Data are shown as normalized fold expressions relative to normal group using β-actin mRNA as internal control. e Representative photomicrographs of immunostaining for MCP-1, ICAM-1, IL-6, and IL-1β in kidney tissue sections taken from anti-Thy1.1 nephritic rats on day 1 after treatment with CLT or CLT-AN. Scale bars , 20 μm. f The levels of MCP-1, ICAM-1, IL-6, and IL-1β were semiquantitatively scored as described in Methods on the basis of immunohistochemical results. In panels a – d and f data are mean ± s.d. ( n = 5), results are representative of two independent experiments. * P < 0.05 vs. PBS group; # P < 0.05 vs. CLT group. Statistical significance was determined by one-way ANOVA with Tukey post hoc test

Article Snippet: After addition of 100 μL Agent A (horseradish peroxidase-conjugated ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, GK500705), the color reaction was performed using 3,3-diaminobenzidine (Sigma-Aldrich, USA), then each section was counterstained with hematoxylin.

Techniques: Real-time Polymerase Chain Reaction, Control, Immunostaining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis

doi: 10.1038/s41467-017-00834-8

Figure Lengend Snippet: Anti-proliferative effects of CLT and CLT-AN in vitro and in vivo. a Effects of CLT and CLT-AN on PDGF-BB-induced proliferation of HBZY-1 cells. b Flow cytometry analysis of the effects of CLT and CLT-AN on the cell cycle distribution of HBZY-1 cells in the presence of PDGF-BB ( C CLT = 0.25 μg mL −1 ). c Representative quadrant plot obtained by flow cytometry analysis showing the ability of CLT and CLT-AN to induce apoptosis in HBZY-1 cells in the presence of PDGF-BB. d Flow cytometry analysis of the proportions of apoptotic HBZY-1 cells after 24-h exposure to CLT or CLT-AN in the presence of PDGF-BB ( C CLT = 0.5 μg mL −1 ). In panels a , b , and d data are mean ± s.d. ( n = 3), results are representative of three independent experiments. * P < 0.05. Statistical significance was determined by one-way ANOVA with Tukey post hoc test. e Real-time PCR analysis of renal mRNA levels of PDGF-BB in anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN. Data are shown as normalized fold expressions relative to normal group using β-actin mRNA as internal control. f Representative photomicrographs of immunostaining for PDGF-BB in kidney tissue sections from anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN, and the levels of PDGF-BB were semiquantitatively scored as described in Methods on the basis of immunochemical results. Scale bars , 20 μm. In panels e , f data are mean ± s.d. ( n = 5), results are representative of two independent experiments. * P < 0.05 vs. PBS group; # P < 0.05 vs. CLT group. Statistical significance was determined by one-way ANOVA with Tukey post hoc test

Article Snippet: After addition of 100 μL Agent A (horseradish peroxidase-conjugated ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, GK500705), the color reaction was performed using 3,3-diaminobenzidine (Sigma-Aldrich, USA), then each section was counterstained with hematoxylin.

Techniques: In Vitro, In Vivo, Flow Cytometry, Real-time Polymerase Chain Reaction, Control, Immunostaining

Journal: Nature Communications

Article Title: Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis

doi: 10.1038/s41467-017-00834-8

Figure Lengend Snippet: Anti-fibrotic effects of CLT and CLT-AN in anti-Thy1.1 nephritic rats. a – d Real-time PCR analysis of renal mRNA levels of Col I ( a ), Col IV ( b ), FN-1 ( c ), TGF-β 1 ( d ) in anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN. Data are shown as normalized fold expressions relative to normal group using β-actin mRNA as internal control. e Representative photomicrographs of immunostaining for TGF-β 1 in kidney tissue sections taken from anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN. Scale bars , 20 μm. f The levels of TGF-β 1 were semiquantitatively scored as described in Methods on the basis of immunochemical results. In panels a – d , f data are mean ± s.d. ( n = 5), results are representative of two independent experiments. * P < 0.05 vs. PBS group; # P < 0.05 vs. CLT group. Statistical significance was determined by one-way ANOVA with Tukey post hoc test

Article Snippet: After addition of 100 μL Agent A (horseradish peroxidase-conjugated ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, GK500705), the color reaction was performed using 3,3-diaminobenzidine (Sigma-Aldrich, USA), then each section was counterstained with hematoxylin.

Techniques: Real-time Polymerase Chain Reaction, Control, Immunostaining