Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: Impacts of overexpression and neutralization of FSTL1 on renal fibrosis and on the canonical Wnt/β-catenin pathways in UUO kidneys. A – C , effects of FSTL1 overexpression on renal fibrosis and on the Wnt/β-catenin pathway. Male mice at 8 weeks of age were subjected to UUO on left ureters. FSTL1-HA or pcDNA3.1 plasmid was injected via the tail vein on day 2 after UUO surgery. Mice were sacrificed the next day, and right (Ctrl) and left (UUO) kidneys were collected for analysis. A , kidney lysates were analyzed by Western blotting for FSTL1 expression and the fibrosis markers α-SMA, fibronectin-1 (Fn-1), and collagen 1 (Col IA1) ( left panel ). Levels of α-SMA, Fn-1, and Col IA1 relative to GAPDH are presented ( right panels ). B , immunofluorescence for α-SMA and Fn-1 and Masson’s trichrome staining (MTS) on sections from the UUO kidneys of mice injected with pcDNA3.1 or FSTL1-HA plasmids. Frozen sections were used for immunofluorescent staining for α-SMA ( green ) and Fn-1 ( red ). Paraffin sections were used for MTS. The fibrotic blue area was quantified. C , kidney lysates were analyzed by Western blotting for the Wnt/β-catenin pathway (p-LRP6, p-GSK3β, active β-catenin, and β-catenin) ( left panel ). Levels of p-LRP6 relative to LRP6; p-GSK3β relative to GSK3β; and active β-catenin and β-catenin relative to GAPDH are presented ( right panels ). D , effects of neutralization of FSTL1 on the Wnt/β-catenin pathway. Male mice at 8 weeks of age were subjected to UUO on left ureters. Normal goat IgG or goat anti-FSTL1 antibody was injected (i.p.) at 5 μg/g body weight 7 days after UUO surgery. About 6 h later, kidney samples were harvested: kidney lysates were analyzed by Western blotting for p-LRP6, active β-catenin, and β-catenin ( left panel ). Levels of p-LRP6 relative to LRP6 and active β-catenin relative to β-catenin and GAPDH are presented ( right panels ). ∗∗ p < 0.01; ∗∗∗ p < 0.001. FSTL1, follistatin-like 1; HA, hemagglutinin; IgG, immunoglobulin G; LRP6, low-density lipoprotein receptor–related protein 6; α-SMA, alpha-smooth muscle actin; TGF-β, transforming growth factor beta; UUO, unilateral ureteral obstruction.

Article Snippet: Membranes were probed with anti-Wnt1 (catalog no.: ab15251; Abcam), anti-FSTL1 (catalog no.: AF1738; R&D Systems), anti-V5 (catalog no.: 13202; Cell Signaling Technology), anti-HA (catalog no.: 05-904; Millipore), anti-WNT3a (catalog no.: 2721; Cell Signaling Technology), anti-Myc (catalog no.: ab9106; Abcam), anti-SNAP (catalog no.: CAB4255; Thermo Fisher Scientific), anti-p-GSK3β (catalog no.: 9336; Cell Signaling Technology), anti-GSK3β (catalog no.: 9315; Cell Signaling Technology), anti-active (nonphospho) β-catenin (catalog no.: 8814; Cell Signaling Technology), anti-β-catenin (catalog no.: 8480; Cell Signaling Technology), anti-p-LRP6 (catalog no.: 2568; Cell Signaling Technology), anti-LRP6 (catalog no.: 3395; Cell Signaling Technology), anti-α-SMA (catalog no.: ab5694; Abcam), anti-Fn (catalog no.: AB2033; Millipore), anti-collagen 1 (catalog no.: bs-10423R; Bioss Antibodies), anti-TGF-β1 (catalog no.: bs-0086R; Bioss), anti-p-Smad3 (catalog no.: ab52903; Abcam), anti-Smad3 (catalog no.: 9523 or 9513; Cell Signaling Technology), anti-p-Smad1/5/8 (catalog no.: 9511; Cell Signaling Technology), anti-Smad1 (catalog no.: 9743; Cell Signaling Technology), anti-p-JNK (catalog no.: 9251; Cell Signaling Technology), anti-JNK (catalog no.: 9252; Cell Signaling Technology), anti-p-CaMKII (catalog no.: 12716; Cell Signaling Technology), and anti-CaMKII (catalog no.: ab52476; Abcam) antibodies.

Techniques: Over Expression, Neutralization, Plasmid Preparation, Injection, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration

doi: 10.1038/s41598-022-18675-x

Figure Lengend Snippet: The histomorphological and immunohistochemical features of the bone-like tissues. ( a ) HE staining showed osteoblast-like cells surrounded by a cohesive osteoid-like matrix and osteocyte-like cells embedded into lacunae-like structures (black arrowheads) in the 3:1, 2:1, and 1:1 co-cultures. ( b , c ) Osteoblast-like cells on the osteoid-like matrix surface were positive for ALP (blue arrowheads) and Col1 (orange arrowheads). ( d ) Osteocyte-like cells in the osteoid-like matrix were positive for DMP1 (green arrowheads). ( e , f ) Fluorescence microscopy showed that osteocyte-like cells embedded in the osteoid-like matrix and osteoblast-like cells on the surface were GFP positive (red arrowheads). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: After a treatment with 0.1% trypsin for 30 min, the sections of bone-like tissues were incubated at 4 °C overnight with a Col1 antibody (NB600-408, 1:100, Novus Biologicals, Littleton, CO, USA) and a DMP1 antibody (sc-54181, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with secondary antibodies.

Techniques: Immunohistochemical staining, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration

doi: 10.1038/s41598-022-18675-x

Figure Lengend Snippet: Transplantation of bone-like tissue in a tooth-extraction mouse model. ( a ) The extraction sockets in the bone-like tissue implantation group showed better healing (red box area). Micro-CT ( b , c ) and HE staining (red box area) ( c ) of the extraction sockets showed that the bone mass in the bone-like tissue implantation group was higher than in unimplanted controls. BV/TV, bone volume/total volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. ( d ) The extraction sockets of the bone-like tissue implantation group had more osteoid and mineralized bone by Goldner’s trichrome staining (red box area). Ordinary white-light microscopy ( e ) and polarized light microscopy ( f ) showed picrosirius red staining for the collagen fibers in the extraction sockets. ( g , h ) The bone-like tissue implantation group had more total collagen and type I collagen (red) than unimplanted controls. * P < 0.05.

Article Snippet: After a treatment with 0.1% trypsin for 30 min, the sections of bone-like tissues were incubated at 4 °C overnight with a Col1 antibody (NB600-408, 1:100, Novus Biologicals, Littleton, CO, USA) and a DMP1 antibody (sc-54181, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with secondary antibodies.

Techniques: Transplantation Assay, Extraction, Micro-CT, Staining, Light Microscopy

Journal: Journal of Clinical Medicine

Article Title: The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype

doi: 10.3390/jcm9040961

Figure Lengend Snippet: Expression of GR, ACTA2 and extracellular proteins in peritubular cells of the monkey testis before and after puberty. Immunohistochemistry of GR of adult rhesus monkeys (right panels) shows strong GR expression in the peritubular compartment (arrows) and moderate staining of Sertoli cells (asterisks). Smooth muscle cells of blood vessels (arrowheads) served as a positive intrinsic control. GR staining of the peritubular wall was not found in all samples from immature rhesus monkeys (186–282 days, left panels and results not shown). Only interstitial cells (asterisks) and blood vessels (arrowheads) show a robust staining. ACTA2 is constantly expressed by vascular smooth muscle cells (arrowheads) in both immature and adult monkeys but only localized in the peritubular wall of mature monkeys (arrows). In immature rhesus, staining of ELN is restricted to the interstitial matrix (asterisks) but is not detected in the peritubular compartment, which in turn is the prevailing expression site in adult monkeys (arrows). Independent of age, Col1 is localized in the peritubular compartment (arrows), in the interstitial area (asterisks), and around blood vessels (arrowheads). Negative control experiments were performed with IgG (not shown). Hematoxylin was used to counterstain nuclei. Scale bars = 50 µm.

Article Snippet: In further IHC experiments, the following primary antibodies were used: anti-ACTA2 (1:200, mouse monoclonal anti-actin, Sigma A5228, St. Louis, MO, USA), Col1 (1:200, affinity-purified, polyclonal rabbit anti-Col1, R1038, Origene, Rockville, MD, USA), and affinity-purified, polyclonal rabbit anti-human ELN (1:200, A5228, Sigma Prestige Antibodies, St. Louis, MO, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Negative Control

Journal: Journal of Clinical Medicine

Article Title: The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype

doi: 10.3390/jcm9040961

Figure Lengend Snippet: Dex treatment of HTPCs rapidly increases FKBP5 mRNA, a known target of GR signaling, and affects mRNA levels of characteristic peritubular ECM markers, as well as cytoskeleton-associated genes. ( A ) Cultured HTPCs stimulated with Dex (1 µM) in the absence or presence of RU486 (1 µM), respond with a highly significantly increase ( p < 0.01) in FKBP5 mRNA levels after 6 h compared to control. RU486 completely blocks this effect ( p < 0.01) and does not alter FKBP5 mRNA expression within 6 h. ( B ) After Dex treatment of HTPCs for 24 h, the mRNAs of the elastic fiber components ELN and FBLN5 are highly significantly ( p < 0.01) increased. A slightly smaller ( p < 0.05) increase is observed for FBN1 mRNA. ( C ) The transcript concentration of the collagen fibers Col1 and Col3 do not change ( p > 0.05) after application of Dex, while ( D ) the mRNAs of cytoskeleton markers ACTA2 and PDLIM1 ( n = 8) are enriched ( p < 0.01) after stimulation of HTPCs with Dex. Data are means ± SEM after 6 ( A ), and 24 h ( B – D ), normalized to control conditions. Asterisks denote statistical significance, * p < 0.05, ** p < 0.01.

Article Snippet: In further IHC experiments, the following primary antibodies were used: anti-ACTA2 (1:200, mouse monoclonal anti-actin, Sigma A5228, St. Louis, MO, USA), Col1 (1:200, affinity-purified, polyclonal rabbit anti-Col1, R1038, Origene, Rockville, MD, USA), and affinity-purified, polyclonal rabbit anti-human ELN (1:200, A5228, Sigma Prestige Antibodies, St. Louis, MO, USA).

Techniques: Cell Culture, Expressing, Concentration Assay