Journal: Kidney international

Article Title: The L1 cell adhesion molecule is a potential biomarker of human distal nephron injury in acute tubular necrosis.

doi: 10.1038/sj.ki.5002640

Figure Lengend Snippet: Figure 4 | Comparative localization of L1 intra- and extracytoplasmic domains. (a) L1 scheme showing the extracellular part with six immunoglobulin-like domains (Ig-like) (including one recognized by mAb272) and five fibronectin III-like (FNIII) domains followed by the transmembrane segment and cytoplasmic fragments (recognized by the goat polyclonal serum). (b–d) Normal CDs are compared with (e–g) damaged CDs. (b, e) CDs are stained by goat polyclonal antiserum labeled by FITC (green) and (c, f) mAb272 labeled by rhodamine (red). (d, g) Merge. (d) In normal CD, both antibodies give a basolateral signal. (g) In injured principal cells, the basolateral signal is low with both antibodies, whereas there is a strong apical membrane (arrow) signal with the anti-cytoplasmic domain antibody contrasting with no signal for the extracellular fragment. Original magnification 630.

Article Snippet: After four washes with TBS-T (Tris-buffered saline with 0.1% Tween), goat anti-human L1 extracellular domain antibody was added (R&D Systems), followed by anti-goat coupled to alkaline phosphatase (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Staining, Labeling, Membrane

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) ATAC-Seq analysis for JUN and the hedgehog genes Gli1 and Ptch1 in scleroderma fibroblasts (SSCL), scleroderma fibroblasts after JUN knockout (JUN-KO) or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( B ) ATAC-Seq analysis for the immune checkpoints CD47 and PD-L1 and the interleukin IL-6 in scleroderma fibroblasts, scleroderma fibroblasts after JUN knockout or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( C ) Heatmap of differential open chromatin regulatory elements characterized from ATAC-Seq. The color bar shows the relative ATAC-Seq signal ( Z score of normalized read counts) as indicated. Samples 1 and 2 in both groups are individual samples. n = 2. ( D ) Fibrosis-linked genes with a 5-fold decline in promoter accessibility after JUN knockout. n = 2. ( E ) p-JUN expression in pulmonary fibroblasts and scleroderma fibroblasts on a 70 kPa hydrogel or a regular polystyrene plastic dish. Two-sided t test. ** P < 0.01; *** P < 0.001. n = 4. Tukey’s multiple comparison test. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Knock-Out, Expressing, Comparison

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Immunofluorescence stains against CD47 and FSP1 with and without JUN induction. Scale bar: 25 μm. n = 5. ( B ) Histogram of CD47 expression in fibroblasts with and without JUN induction. n = 5. ( C ) Percentage of CD47 positivity in different fibroblast populations with and without JUN induction. Fisher’s multiple comparison test. ** P < 0.01; *** P < 0.01. n = 5. Bars represent means with standard deviations. ( D ) Representative optical images of ectopically transplanted JUN-inducible mouse dermal fibroblasts ± CD47 inhibition. n = 4. ( E ) Corresponding quantification of photon emissions. Values are normalized to day 0. Fisher’s multiple comparison test. ** P < 0.01. n = 4. Bars represent means with standard deviations. ( F ) Fluorescent graft visualization under the dissection microscope after 7 days of CD47 inhibition. Scale bar: 5 mm. n = 2. ( G ) FACS plot for PE/RFP + CD11b + macrophages ± JUN induction ± CD47 inhibition in an in vitro phagocytosis assay. n = 3. ( H ) Corresponding quantification of RFP + macrophages. Tukey’s multiple comparison test. * P < 0.05; *** P < 0.01. n = 3. Bars represent means with standard deviations. ( I ) Schema of a macrophage depletion trial with subsequent skin fibrosis induction. n = 5. ( J ) Corresponding trichrome stains. Scale bar: 500 μm. n = 5. ( K ) Corresponding hydroxyproline assay. Two-sided t test. *** P < 0.001. n = 5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Immunofluorescence, Expressing, Comparison, Inhibition, Dissection, Microscopy, In Vitro, Phagocytosis Assay, Hydroxyproline Assay

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Schematic outline of the therapeutic trial. ( B ) Representative H&E and trichrome stains of the different groups. Scale bar: 500 μm. n = 4. ( C ) Hydroxyproline content of the skin. Tukey’s multiple comparison test. * P < 0.05; ** P < 0.01. n = 6. Graph bars represent means with standard deviations. ( D ) Amount of fat tissue. Values indicate μm 2 /μm skin width. Tukey’s multiple comparison test. * P < 0.05. n = 8. Graph bars represent means with standard deviations. ( E ) Representative optical images of ectopically transplanted GFP/luciferase-labeled human scleroderma fibroblasts ± CD47/IL-6 inhibition. ( F ) Optical imaging of explanted kidneys on day 5. ( G ) Quantification of photon emissions of explanted kidney grafts normalized to the values of the untreated mice. Two-sided t test. * P < 0.05. n = 3–4. Bars represent means with standard deviations. ( H ) Corresponding caspase-3 staining of kidney grafts. Scale bar: 25 μm. ( I ) Corresponding percentage of caspase-3 + GFP + fibroblasts. Two-sided t test. ** P < 0.01. n = 4–5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Comparison, Luciferase, Labeling, Inhibition, Optical Imaging, Staining