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

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