Journal: The Journal of Clinical Investigation

Article Title: ADAMTS7 promotes smooth muscle foam cell expansion in atherosclerosis

doi: 10.1172/JCI187451

Figure Lengend Snippet: ( A ) Schematic outlining experimental design and the generation of the SMC transgenic ADAMTS7 mouse. Created with BioRender.com. ( B ) ORO staining of the en face aorta and quantification of lesion area. n = 9. ( C ) Representative images of H&E-stained aortic root sections, quantification of plaque areas ( n = 6). Scale bar: 500 μm. ( D ) Representative images of H&E-stained aortic root sections with necrotic core outlined in dotted line ( n = 6 mice). Scale bar: 300 μm. ( E ) Representative images of aortic root sections stained against α-SMA (Cy3; red), MAC2 (Alexa Fluor 488 green), and cell nuclei (DAPI blue) with their subsequent quantification relative to lesion area ( n = 6). Scale bar: 300 μm. ( F ) Representative Picrosirius red staining of aortic root lesions with bars indicating fibrous cap thickness and quantification of fibrous cap thickness ( n = 6). Scale bar: 300 μm. ( G ) Schematic outlining experimental design and the generation of the EC transgenic ADAMTS7 mouse. Created with BioRender.com. ( H ) ORO staining of the en face aorta and quantification of lesion area. n = 3–4. ( I ) Representative images of H&E-stained aortic root sections and quantification of plaque areas ( n = 5–6). Scale bar: 500 μm. **** P < 0.0001, * P < 0.05. Statistics were analyzed using a 2-tailed Student’s t test.

Article Snippet: The sections then were stained using the following Abs and dilutions: 1:1,000 α-SMA-Cy3 (MilliporeSigma, C6198) and 1:500 MAC2 (Cedarlane, CL8942AP).

Techniques: Transgenic Assay, Staining

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining

Journal: Science Advances

Article Title: A long noncoding RNA promotes parasite differentiation in African trypanosomes

doi: 10.1126/sciadv.abn2706

Figure Lengend Snippet: ( A ) Pipeline used for the identification of lncRNAs genes in T. brucei . (1) Strand-specific and paired-end RNA-seq. (2) Ksplice identified putative genes whose transcripts contained an SL sequence (SL) and a poly(A) tail (PA) at the extremities. Ksplice used LAST to map RNA-seq reads to the T. brucei genome. (3) The noncoding nature of the putative lncRNAs was predicted from a low coding potential calculator (CPC) score, poor association with ribosomes, and no detectable peptides. grumpy lncRNA is intergenic and immediately upstream of RBP7 genes, previously shown to be involved in the SIF-dependent pathway. ( B ) The number of full-length lncRNAs [from the SL sequence to the poly(A) tail] sequenced with Nanopore in four RNA samples: two from BSF parasites (BSF) and two from procyclic forms (PCF). ( C ) Distribution of poly(A) tail lengths in lncRNA candidates versus other transcripts. ( D ) Subcellular localization of Ksplice lncRNA genes in slender forms (SL), stumpy forms (ST), and PCF of T. brucei , using RNA-FISH. ( E ) The number of Ksplice lncRNA genes that cause loss of parasite fitness upon down-regulation by RNAi [extracted from RIT-seq analysis ]. RNAi was induced in BSFs for 3 days (BSD3), 278 lncRNAs; in BSFs for 6 days (BSD6), 341 lncRNAs; during in vitro parasite differentiation from BSF to insect procyclic forms (DIF), 400 lncRNAs; in PCF, 402 lncRNAs. The total number of lncRNA genes essential for parasite fitness in this screen was 649.

Article Snippet: Parasite differentiation into procyclic forms was assessed at 12 hours after differentiation by flow cytometry using anti– T. brucei procyclin antibody (0.5 mg; Cedarlane, clone TBRP1/247, CLP001AP) conjugated with Alexa Fluor 647 (protein labeling kit, Molecular Probes) (1:500 dilution in TDB).

Techniques: RNA Sequencing, Sequencing, In Vitro

Journal: Science Advances

Article Title: A long noncoding RNA promotes parasite differentiation in African trypanosomes

doi: 10.1126/sciadv.abn2706

Figure Lengend Snippet: ( A ) Transcript-level changes during the transition from slender to stumpy forms, measured by RT-qPCR. Stumpy formation was induced by pCPT-cAMP (C3912, Sigma-Aldrich). Tb927.2.2220 is used as a control to normalize transcript levels . PAD1 and GFP genes are used as controls to estimate parasite differentiation into stumpy forms. Tb927.10.12080 is the gene upstream of grumpy . Results are shown as means (SEM, n = 3 except for the 48-hour time point, n = 2). Dunnett’s multiple comparisons test was used for statistical analysis using the 0-hour time point as the control for comparison (adjusted P values: * P < 0.05; ** P < 0.01; **** P < 0.0001). ( B ) Subcellular localization of grumpy during the transition from slender to stumpy forms using RNA-FISH. Time points (0, 12, 24, 36, and 48 hours) of parasite differentiation after addition of the pCPT-cAMP stimulus to the culture medium. DIC, differential interference contrast microscopy image of T. brucei ; GFP, GFP::PAD1 signal expressed in the nucleus of stumpy forms; GRUMPY , grumpy signal using RNA-FISH (Stellaris probes). ( C ) Quantification of the intensity of the nucleolar grumpy RNA-FISH signal corrected with the cytoplasmic signal, during stumpy formation induced by pCPT-cAMP at time 0, 24, and 48 hours. Statistical test: one-way analysis of variance (ANOVA) (Dunnett’s multiple comparisons test), adjusted P value: **** P < 0.0001.

Article Snippet: Parasite differentiation into procyclic forms was assessed at 12 hours after differentiation by flow cytometry using anti– T. brucei procyclin antibody (0.5 mg; Cedarlane, clone TBRP1/247, CLP001AP) conjugated with Alexa Fluor 647 (protein labeling kit, Molecular Probes) (1:500 dilution in TDB).

Techniques: Quantitative RT-PCR, Control, Comparison, Microscopy

Journal: Science Advances

Article Title: A long noncoding RNA promotes parasite differentiation in African trypanosomes

doi: 10.1126/sciadv.abn2706

Figure Lengend Snippet: ( A ) Transcript levels measured by RT-qPCR of grumpy and its neighboring genes in parental cell line (black bars) and in grumpy- overexpressing (OE) cell line, 24 hours after tetracycline induction (yellow bars). Changes in transcript levels were measured by normalizing transcript level to a control gene (Tb927.10.12970) and to the parental cell line. ( B ) Subcellular localization of grumpy after overexpression using RNA-FISH. Left to right: GRUMPY , grumpy signal using RNA-FISH; phase-contrast signal of T. brucei parasite; GFP, GFP::PAD1 signal expressed in the nucleus of stumpy forms. ( C to F ) After inducing grumpy overexpression, we measured (C) parasite growth for 6 days (without passage), (D) percentage of live cells measured by fluorescence-activated cell sorting (FACS) of propidium iodide–stained cells, (E) percentage of GFP::PAD1-positive parasites (stumpy forms) measured by FACS, (F) percentage of parasites expressing both GFP::PAD1 and endogenous PAD1 protein that are measured by microscopy and image quantification. Microscopy picture (on the left) shows an example of a parasite expressing both GFP::PAD1 in the nucleus (in green) and the endogenous PAD1 protein at the cell surface (in red). Parasite DNA stained with 4′,6-diamidino-2-phenylindole (DAPI) (in blue). ( G ) Cell cycle profile of parental cell line (slender forms) and grumpy -overexpressing parasites at days 3 and 4 of in vitro culture without passage. All results are shown as means (SEM, n = 3). Multiple t tests (A) and Sidak’s multiple comparisons test (C to G) are used for statistical analysis using the parental cell line as the control for comparison (adjusted P values: * P <0.05; ** P < 0.01; *** P <0.001; **** P < 0.0001).

Article Snippet: Parasite differentiation into procyclic forms was assessed at 12 hours after differentiation by flow cytometry using anti– T. brucei procyclin antibody (0.5 mg; Cedarlane, clone TBRP1/247, CLP001AP) conjugated with Alexa Fluor 647 (protein labeling kit, Molecular Probes) (1:500 dilution in TDB).

Techniques: Quantitative RT-PCR, Control, Over Expression, Fluorescence, FACS, Staining, Expressing, Microscopy, In Vitro, Comparison