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: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Rasip1 expression in IAHCs in the E10.5 AGM region. A . Upper panel: The expression profile of CD45, c-Kit, CD31, and VE-Cad in the E10.5 AGM region. The numbers from 1 to 4 indicate cell populations separated by the expression profile: 1.CD45 − c-Kit −- CD31 + VE-cad − cells (endothelial cells); 2.CD45 − c-Kit − CD31 + VE-cad + cells (endothelial cells); 3.CD45 low c-Kit high cells (containing HSPCs); 4.CD45 + c-Kit − cells (hematopoietic cells). Lower panel: RT-PCR shows the relative expression levels of Rasip1 in these 4 populations. B . Whole-mount immunofluorescence staining of Rasip1 (green), CD31 (red), and c-Kit (cyan) in IAHCs of the E10.5 AGM region. The Rasip1 expression on the membrane of IAHCs. The nuclei were stained by Hoechst 33258 (blue). Scale bar, 20 μm. C . A genetic scheme showing the location of the Rasip1 gene and upstream genes in mouse chromosome 7. 5 putative Sox17-binding sites were indicated by squares with numbers 1 through 5. D . Upper panel: Requirement of the proximal putative Sox17-binding site (5) for induction of the Rasip1 gene by Sox17. NIH3T3 cells (1 × 10 6 ) were transfected with a pGL3 vector containing the putative Rasip1 promoter sequences. pRL-CMV was co- transfected as an internal control. The region Rasip1 [ - ] (979 bp) has three putative Sox17- binding sites (1, 2, and 3), and the region Rasip1 [ - ] (602 bp) has two putative Sox17 binding sites (2 and 3). The region Rasip1 [ - ] (720 bp) has putative Sox17-binding sites 4 and 5. Two types of Sox17-binding sites are shown in the promoter regions: 1. AATGGCG (Sox17 binding sites 2, 4); 2. ATTGT (Sox17 binding sites 1,3,5). Lower panel: Inhibition of the Sox17-induced Rasip1 gene activation by a mutation in the putative Sox17-binding site 5. NIH3T3 cells (1 × 10 6 ) were transfected with a pGL3 vector containing the putative Rasip1 promoter sequences with point mutations in the putative Sox17-binding sites 4 or 5, or both together with plasmids encoding wild-type (WT) Sox17 or Sox17G103R which lost the DNA binding ability. In this experiment, pRL-TK was co-transfected as an internal control. A solid black line indicates the Rasip1 [ - ] region, solid grey lines show the pGL3 vector and Rasip1 promoter region [ - ] as controls.

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Binding Assay, Transfection, Plasmid Preparation, Inhibition, Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Expression of Rasip1 in Sox17 (GFP)-transduced CD45 low c-Kit high cells. A . Upper panel: A schema of Sox17-IRES-GFP (Sox17) and IRES-GFP (Mock) retrovirus vectors. Lower panel: The red squares show a region of the CD45 low c-Kit high cell population. The expression of Rasip1 in Mock- or Sox17-transduced cells was analyzed by RT-PCR and normalized by β-actin. B . Upper panel: A schema of the Sox17-ERT-IRES-GFP retrovirus vector. Lower panel: The dark green squares show Sox17 (GFP) + CD45 low c-Kit high cells. After FACS sorting, expression analysis of the Rasip1 gene was performed in Sox17-ERT- IRES-GFP-transduced cells with or without tamoxifen (1.0 μg/ml).

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Rasip1 knockdown (KD) in CD45 low c-Kit high cells. A . Upper panel: A schema of the position of the Rasip1 shRNA in the Rasip1 mRNA, a retrovirus vector of Sox17-IRES- mCherry, and retrovirus vectors of shLuc and shRasip1. Lower panel: After 4 days of Sox17- IRES-mCherry transduction, Sox17-transduced cells formed cell clusters. shLuc and shRasip1 were introduced into Sox17-transduced cells and the cells were co-cultured with OP9 stromal cells. Morphologies of shRasip1 (green, GFP + )-transduced Sox17 + cells (red, mCherry + ). Scale bar, 1.0 mm. B . shLuc- or shRasip1-transduced GFP + cells were recovered by FACS. Expression of Rasip1 in shLuc- and shRasip1-transduced cells was analyzed by RT-PCR and normalized by β-actin gene expression. RT-PCR result is in the static image that is generated by a mirror-reversal of an original across a horizontal axis, due to the sample order in the electrophoresis. C . Sorted GFP + cells (2.5 × 10 3 ) were embedded in a semi-solid medium. The number of total (CFU-C) and mix (CFU-Mix) colonies were scored after 7 days of culture. The pair of colony numbers (sh-Luc and shRasip1) in each experimental group were plotted and connected by a line. The different marks indicate the different individual embryos (n = 7, 7 individual liters of mice from which each experimental group was set up).*p≤0.05; **p≤0.01; ***p≤0.005.

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: shRNA, Plasmid Preparation, Transduction, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Electrophoresis

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Rasip1 overexpression (OE) in CD45 low c-Kit high cells. A . A schema of IRES-GFP (Mock) and Rasip1-IRES-GFP retrovirus vectors. B . Timeline of Rasip1 OE experiment. C . Red squares show Mock- and Rasip1-transduced CD45 low c-Kit high cells. D . After 4 days of Mock and Rasip1 transduction, CD45 low c-Kit high cells (2.5 × 10 2 ) were embedded in a semi- solid medium (Colony assay 1). According to the timeline in , CD45 low c-Kit high cells were sorted (5.0 × 10 2 ) after 8 days of Rasip1 transduction (Colony assay 2). The number of total (CFU-C) and multilineage (CFU-Mix) colonies were scored after 7 days of culture (n=5). The pair of colony numbers (Mock and Rasip1) in each experimental group were plotted and connected by a line. The different marks indicate the different individual embryos (n=6, 6 individual liters of mice from which each experimental group was set up). *p≤0.05; **p≤0.01.

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Over Expression, Transduction, Colony Assay

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

Article Title: MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias

doi: 10.1073/pnas.1816177115

Figure Lengend Snippet: MIMEX15-mutant Purkinje neurons undergo autophagy. (A) MIMEX15 mutants display fused mitochondria shown by increased complex 5 ATP-synthase immunostaining and collapsed Golgi shown by reduced giantin immunostaining at 4 wk. (B) Eight-week-old MIMEX15 mutants show increased LC3-II abundance. *P < 0.005; Student’s t test. (C) MIMEX15 mutants show increased levels of mRNA for the autophagocytic marker VMP1. *P < 0.05; Student’s t test. (D) MIMEX15 mutants show increased microglial infiltration shown by Aif1 transcript. *P < 0.002; Student’s t test. (E) MIMEX15 mutants show GFAP+ glial infiltration during disease progression. (F) Western blots quantifying increased cerebellar GFAP. *P < 0.05; Student’s t test. (G) MIMEX15-mutant cerebella do not have increased TUNEL staining at 4, 8, or 16 wk of age.

Article Snippet: The following antibodies were used at 1:1,000 dilutions: rabbit anti-Mtss1 ( 30 ), rabbit anti-calbindin (13176; Cell Signaling Technology ), mouse anti–calbindin-D-28K monoclonal (Sigma), mouse anti-complex V (Novex; 459240), rabbit anti-ubiquitin (3933; Cell Signaling Technology), rabbit anti-giantin (Abcam; ab24586), and chicken anti-GFAP (ab4674; Abcam).

Techniques: Mutagenesis, Immunostaining, Marker, Western Blot, TUNEL Assay, Staining

Journal: Oncogene

Article Title: Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth

doi: 10.1038/s41388-020-01523-5

Figure Lengend Snippet: A Western blotting showing the expression of p-STAT3 and STAT3 in healthy colon or colon tumors. B Densitometry quantification of p-STAT3/total STAT3 for colons or colorectal tumors. *** p < 0.001 (one-way ANOVA). C Representative images of p-STAT3 immunostaining of colons or colon tumors. Scale bar: 50 μm. D Quantification of p-STAT3 + cells in colon tumors of WT and Gal2-KO mice. *** p < 0.001 ( t -test).

Article Snippet: Primary antibodies including the goat polyclonal anti-Gal2 (STJ24400, 1:500, St John’s Laboratory Ltd, London, UK), anti-STAT3 (#9139, 1:1000), anti-p-STAT3 (#9145, 1:1000), and anti-GAPDH (MAB374, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Immunostaining

Journal: Oncogene

Article Title: Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth

doi: 10.1038/s41388-020-01523-5

Figure Lengend Snippet: A Western blotting showing the expression of Gal2 in stable Gal2-overexpressing HCT116 cells. The red arrow indicates the un-cleaved GFP-2A-Gal2 fusion protein. B The CCK8 proliferation assay of HCT116 with or without Gal2 overexpression. C The survival assay of HCT116 with or without Gal2 overexpression in the presence of 0.6 mM H 2 O 2 . ** p < 0.01, *** p < 0.001 ( t -test). D Western blotting showing the expression of p-STAT3 and STAT3 in HCT116 cells with or without Gal2 overexpression. E Densitometry quantification of p-STAT3/total STAT3 in HCT116 cells with or without Gal2 overexpression. ** p < 0.01 (one-way ANOVA).

Article Snippet: Primary antibodies including the goat polyclonal anti-Gal2 (STJ24400, 1:500, St John’s Laboratory Ltd, London, UK), anti-STAT3 (#9139, 1:1000), anti-p-STAT3 (#9145, 1:1000), and anti-GAPDH (MAB374, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Proliferation Assay, Over Expression, Clonogenic Cell Survival Assay