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: Frontiers in Immunology

Article Title: A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis

doi: 10.3389/fimmu.2022.860807

Figure Lengend Snippet: Information for immunofluorescence antibodies and other reagents.

Article Snippet: Primary antibodies were anti-TGF-β RI (bs-0638R, Bioss, China), anti-GAPDH (5174S, Cell Signaling Technology, CST, USA), anti-α-SMA (19245S, CST), anti-Col 1 α1 (bs-7158R, Bioss), anti-Col 3 α1 (22734-1-AP, Proteintech, USA), anti-Smad3 (9523S, CST), anti-phospho-Smad3 (9520S, CST), anti-Flotillin-1 (3253S, CST), and anti-HSP70 (46477S, CST).

Techniques: Immunofluorescence

Journal: Frontiers in Immunology

Article Title: A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis

doi: 10.3389/fimmu.2022.860807

Figure Lengend Snippet: Primer sequences.

Article Snippet: Primary antibodies were anti-TGF-β RI (bs-0638R, Bioss, China), anti-GAPDH (5174S, Cell Signaling Technology, CST, USA), anti-α-SMA (19245S, CST), anti-Col 1 α1 (bs-7158R, Bioss), anti-Col 3 α1 (22734-1-AP, Proteintech, USA), anti-Smad3 (9523S, CST), anti-phospho-Smad3 (9520S, CST), anti-Flotillin-1 (3253S, CST), and anti-HSP70 (46477S, CST).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis

doi: 10.3389/fimmu.2022.860807

Figure Lengend Snippet: Egg-derived exosomes from Schistosoma japonicum can promote liver fibrosis. (A) Western blotting detection of markers of egg-derived exosomes of S. japonicum . (B) Transmission electron microscopy (TEM) analysis of exocrine. (C) At 24 h after stimulation of LX-2 human hepatic stellate) cells (HSCs) by egg-derived exosomes of S. japonicum , mRNA levels of smooth muscle actin (α-SMA), collagen (Col) 1 (α1) , and collagen (Col) 3 (α1) were measured by qPCR. Results are averaged from three independent experiments, and Student’s t -tests were applied to assess differences between two groups. The mRNA expression levels of α-SMA , Col 1 (α1) and Col 3 (α1) were normalized against GAPDH ** p < 0.001, *** p < 0.0005.

Article Snippet: Primary antibodies were anti-TGF-β RI (bs-0638R, Bioss, China), anti-GAPDH (5174S, Cell Signaling Technology, CST, USA), anti-α-SMA (19245S, CST), anti-Col 1 α1 (bs-7158R, Bioss), anti-Col 3 α1 (22734-1-AP, Proteintech, USA), anti-Smad3 (9523S, CST), anti-phospho-Smad3 (9520S, CST), anti-Flotillin-1 (3253S, CST), and anti-HSP70 (46477S, CST).

Techniques: Derivative Assay, Western Blot, Transmission Assay, Electron Microscopy, Expressing

Journal: Frontiers in Immunology

Article Title: A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis

doi: 10.3389/fimmu.2022.860807

Figure Lengend Snippet: Novel miRNA-33 mimics can activate HSCs (human hepatic stellate cells). (A) LX-2 cells were transfected with several novel miRNA mimics (novel miRNA-30, novel miRNA-33, novel miRNA-68, miR-1, miRNA-NC), cultivated for 24 h, and mRNA levels of α-SMA and Col 1 (α1) were measured by qPCR. (B) LX-2 cells were transfected with several novel miRNA mimics (novel miRNA-30, novel miRNA-33, novel miRNA-68, miR-1, miRNA-NC), cultivated for 48 h, and expression levels of α-SMA and Col 1 (α1) were measured by western blotting. Results were compared with those of the NC mimic group. The abundances of α-SMA and Col 1 (α1) proteins were normalized against GAPDH. Results are averaged from three independent experiments, and Student’s t -tests were applied to assess differences between two groups (** p < 0.001, **** p < 0.0001).

Article Snippet: Primary antibodies were anti-TGF-β RI (bs-0638R, Bioss, China), anti-GAPDH (5174S, Cell Signaling Technology, CST, USA), anti-α-SMA (19245S, CST), anti-Col 1 α1 (bs-7158R, Bioss), anti-Col 3 α1 (22734-1-AP, Proteintech, USA), anti-Smad3 (9523S, CST), anti-phospho-Smad3 (9520S, CST), anti-Flotillin-1 (3253S, CST), and anti-HSP70 (46477S, CST).

Techniques: Transfection, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis

doi: 10.3389/fimmu.2022.860807

Figure Lengend Snippet: Novel miRNA-33 agomir causes liver fibrosis in mice. Six-week-old female C57BL/6 mice were injected with agomir NC (Ribo), agomir novel 33 (Ribo), or normal saline once a week for 6 weeks. Results were compared with the agomir NC group; the normal group was only used as a reference without comparative analysis. Only the gray values of the agomir NC group and the agomir novel 33 group were statistically analyzed, the blank group was not analyzed. (A) Novel miRNA-33 levels in mouse liver and serum were increased significantly. (B) qPCR results showing upregulation of α-SMA, Col 1 (α1) , and Col 3 (α1) at the mRNA level. (C) Western blotting results showing upregulation of α-SMA, Col 1 (α1), and Col 3 (α1) at the protein level. The abundances of α-SMA, Col 1 (α1), and Col 3 (α1) proteins were normalized against GAPDH, and miRNA expression levels were normalized against U6. Results are averaged from three independent experiments, and Student’s t -tests were applied to assess differences between two groups (** p < 0.001, *** p < 0.0005, **** p < 0.0001).

Article Snippet: Primary antibodies were anti-TGF-β RI (bs-0638R, Bioss, China), anti-GAPDH (5174S, Cell Signaling Technology, CST, USA), anti-α-SMA (19245S, CST), anti-Col 1 α1 (bs-7158R, Bioss), anti-Col 3 α1 (22734-1-AP, Proteintech, USA), anti-Smad3 (9523S, CST), anti-phospho-Smad3 (9520S, CST), anti-Flotillin-1 (3253S, CST), and anti-HSP70 (46477S, CST).

Techniques: Injection, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis

doi: 10.3389/fimmu.2022.860807

Figure Lengend Snippet: Inhibition of novel miRNA-33 reduces the degree of liver fibrosis. Six-week-old female C57BL/6 mice were percutaneously exposed to 20 ± 1 S. japonicum cercariae, and 120 μL of 20 nM miRNA antagomir NC(Ribo), miRNA antagomir novel 33 (Ribo), or normal saline were injected via the tail vein 7 days after mice were infected with S. japonicum cercariae, once a week for 6 weeks. The magnification of Masson-stained images is 10 ×, and the magnification of immunofluorescence image is 20 ×. (A, B) Masson staining showing that areas of collagen were decreased after treatment with novel miRNA-33 antagomir. (C) Egg count in mouse liver tissue showing there was no statistical difference between the three groups. (D) Expression of novel miRNA-33 in mice infected with S. japonicum for 6 weeks. (E) qPCR results showing downregulation of α-SMA, Col 1 (α1), and Col 3 (α1) at the mRNA level. (F) Immunofluorescence images showing downregulation of type I collagen and α-SMA after treatment with antagomir. mRNA expression levels of α-SMA , Col 1 (α1) , and Col 3 (α1) were normalized agaisnt GAPDH , and expression of novel miRNA-33 was normalized against U6. Results are averaged from three independent experiments, Student’s t -tests were applied to assess differences between two groups, and one-way analysis of variance (ANOVA) followed by Tukey’s post-tests was used for three or more groups (fluorescence is SpGreen; n = 20; * p < 0.05, ** p < 0.001, *** p < 0.0005; NS, not significant).

Article Snippet: Primary antibodies were anti-TGF-β RI (bs-0638R, Bioss, China), anti-GAPDH (5174S, Cell Signaling Technology, CST, USA), anti-α-SMA (19245S, CST), anti-Col 1 α1 (bs-7158R, Bioss), anti-Col 3 α1 (22734-1-AP, Proteintech, USA), anti-Smad3 (9523S, CST), anti-phospho-Smad3 (9520S, CST), anti-Flotillin-1 (3253S, CST), and anti-HSP70 (46477S, CST).

Techniques: Inhibition, Injection, Infection, Staining, Immunofluorescence, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Melatonin Exerts Prominent, Differential Epidermal and Dermal Anti-Aging Properties in Aged Human Eyelid Skin Ex Vivo

doi: 10.3390/ijms242115963

Figure Lengend Snippet: List of antibodies and immunohistochemical staining treatments used in the study.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-phospho-S6 ribosomal protein (Ser235/236) (1:200; Cell Signaling, Danvers, MA, USA, #4858), rabbit monoclonal anti-COL17A1 [EPR14758] (1:200; Abcam, Cambridge, MA, USA, #ab186415), mouse monoclonal anti-MMP1 (1:100; Biolegend, San Diego, CA, USA, #634702), rabbit monoclonal anti-TFAM [EPR12285](1:500; Abcam, #ab176558), rabbit monoclonal anti-VDAC1/Porin (1:100; Abcam, #ab15895), rabbit monoclonal anti-MTCO1 [EPR19628] (1:50; Abcam, #ab203912), rabbit monoclonal anti-human Alexa Fluor ® 488 Anti-VEGFA [EP1176Y] (1:500, Abcam, #ab206886), rabbit monoclonal anti-SIRT1 [E104] (1:200; Abcam, #ab32441), rabbit monoclonal anti-lamin B1 [EPR8985(B)] (1:100; Abcam, #ab194109), rabbit monoclonal anti-phospho-histone γH2A.X (Ser139) (1:1500; Cell Signaling, #2577S), rabbit monoclonal anti-CDKN2A/p16 INK4a [EPR1473] (1:250; Abcam, #ab108349), mouse monoclonal anti-fibrillin-1 biotinylated [11C1.3] (1:800; Abcam, #ab24826), and mouse monoclonal anti-Collagen I(1:500; Novus Bio, Centennial, CO, USA, #NB600-450).

Techniques: Immunohistochemical staining, Staining, Blocking Assay, Amplification, Avidin-Biotin Assay