Journal: JCI Insight

Article Title: TGF- β promotes fibrosis after severe acute kidney injury by enhancing renal macrophage infiltration

doi: 10.1172/jci.insight.123563

Figure Lengend Snippet: Peritoneal macrophages from Tgfbr2fl/fl (WT) and LysM-Cre Tgfbr2fl/fl (macrophage TGF-βRII–/–; MφTGF-βRII–/–) mice 3 days after peritoneal thioglycollate administration were used. (A) TGF-βRII levels were markedly lower in TGF-βRII–/– macrophages than in WT macrophages. ***P < 0.001, n = 3 in each group. Data were expressed as mean ± SEM. (B) Macrophages were stimulated with 2 ng/ml recombinant TGF-β for 30 minutes. TGF-βRII deletion led to decreases in TGF-β–stimulated phosphorylation of Smad2 and Smad3, an indication of TGF-βRII deficiency. p-Smad3, phospho-Smad3; p-Smad2, phospho-Smad2; FAK, focal adhesion kinase.

Article Snippet: Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) and CD3 (MCA1477, a marker of T lymphocytes) were purchased from AbD Serotec (now Bio-Rad); mouse anti–mannose receptor (CD206, MAB25341) and mouse anti–4-HNE (a marker of oxidative stress, 198960) were from R&D Systems; rabbit anti–TGF-βRII (SC-400) and goat anti-human CTGF (SC-14939) were from Santa Cruz Biotechnology; rabbit anti-human Smad2 (600-401-A59), phospho-Smad2 (600-401-K09S), and phospho-Smad3 (600-401-919) as well as rabbit anti-murine collagen type I (600-401-103-01) were from Rockland Immunochemicals; mouse anti–α-SMA (A5228, a marker of myofibroblasts) was from Sigma-Aldrich; and rabbit anti–TGF-β1 (NBP1-80289) and mouse anti–TGF-β2 (Mab612-SP) were from Novus Biologicals.

Techniques: Recombinant

Journal: Nature Communications

Article Title: High-fat diet disturbs lipid raft/TGF-β signaling-mediated maintenance of hematopoietic stem cells in mouse bone marrow

doi: 10.1038/s41467-018-08228-0

Figure Lengend Snippet: Short-term HFD disturbs lipid raft/TGF-β signaling on HSC. a LR hi HSC shows important phosphorylation of Smad2/3 (pSmad2/3) in CD-fed mice, and a HFD affects pSmad2/3 expression among HSC. The pSmad2/3 and LR (CTB) are detected by flow cytometry and results show the distribution (%) of pSmad2/3 + /LR hi and pSmad2/3 − /LR lo cells gated on LSK-CD34 − HSC, n = 5 mice per diet group. b Single-cell immunostaining is performed on LSK-CD34 − LR hi cells isolated by FACS. Data show clustering of LR (CTB) when mice are fed a HFD, n = 4 mice per diet group. c Single-cell immunostaining on LSK-CD34 − LR hi cells showing that HFD induces cluster formation of LR (CTB, red), in which Tgfbr1 (green) is typically condensed. Data show examples of representative single-cell immunostaining for CD and HFD-fed mice, n = 4 mice per diet group. Statistics of the colocalization on LSK-CD34 − cells ( n > 30 cells) is shown on the right panel. Microscopy is performed on LSK-CD34 − LR hi cells isolated by FACS. White scale bars represent 5 µm. a , b Data show mean ± SD. c Data are presented as median (central line), first and third quartiles (bottom and top of boxes, respectively), and whiskers (extreme values); * P < 0.05; *** P < 0.001 (two-tailed unpaired Student’s t -test)

Article Snippet: For the phospho-Smad2/3 microscopy, cells were stained with the cell surface markers (LSK-CD34 − ) and AF555-conjugated CTB (C-34776, Thermo Fisher Scientific, 1 µg/mL), prior to fixation/permeabilization using BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences), then we stained with an anti-phospho-Smad2(S465/S467) antibody (AB3849, Merck, dilution ratio 1:250) and with secondary anti-rabbit AF488 (A27034, Thermo Fisher Scientific, dilution ratio 1:1000) antibody.

Techniques: Expressing, Flow Cytometry, Immunostaining, Isolation, Microscopy, Two Tailed Test