Journal: bioRxiv

Article Title: Microsecond pulse electrical stimulation modulates cell migration

doi: 10.1101/2022.10.23.513372

Figure Lengend Snippet: (a) A graphical illustration showed the effects of μsPEF (pulse width:20 μs, frequency: 10 Hz, duration: 5 s) on the skin wound, promoting cell migration and extracellular matrix remodeling. (b) Representative time-lapse images showing the fibroblasts morphology after μsPEF (i.e., 750 and 1500 V/cm) treatment of different intensity at different time points (i.e., 0, 1 and 2 h). Detached cells are marked by yellow arrow. Scale bar, 50 μm. (c) The line graph representing the cell migration average speed per 1 h from 0 to 12 h after different intensity μsPEF (i.e., 750 and 1500 V/cm) treatment (blue circle: control; orange square: 750 V/cm; pink triangle: 1500 V/cm). Results are presented as mean ± standard deviation with 95% CI (n CTRL =42 cells, n 750 v/cm =48 cells, n 1500 v/cm =47 cells); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons. (d) Box plot showing the average cell migration speed with different intensity μsPEF (i.e., 750 and 1500 V/cm) and control treatments in 24 h. Results are presented as mean ± standard deviation with 95% CI (n CTRL =470 cells, n 750 V/cm =979 cells, n 1500 V/cm =535 cells). (e) Effects of μsPEF on the secretion of COLA2. The level of collagen type I α2 in cellular supernatants was measured after 48 h. The concentration of COLA2 was 0.109 ± 0.018 ng/mL in the control group, 0.257 ± 0.058 ng/mL in the 750 V/cm group and 0.363 ± 0.034 ng/mL in the 1500 V/cm group. Results are presented as mean ± standard deviation with 95% CI (n=5); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons. (f) Effects of μsPEF on the expression of FGF2. The level of FGF2 in cellular supernatants was measured after 48 h. The concentration of FGF2 was 44.139 ± 0.360 ng/mL in the control group, 48.012 ± 1.488 ng/mL in the 750 V/cm group and 48.523 ± 1.944 ng/mL in the 1500 V/cm group. Results are presented as mean ± standard deviation with 95% CI (n=3); ns=0.7329, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons.

Article Snippet: Then, the cells were cultured in serum-free medium for 48 h. The content of type I α collagen and basic fibroblast growth factor (FGF-2) in the supernatant were measured using commercially available Human COL1A2 (Collagen Type I Alpha 2) ELISA Kit (Elabscience, Wuhan, China) and Human bFGF/FGF2 (Basic Fibroblast Growth Factor) ELISA Kit (Elabscience, Wuhan, China) according to the manufacturer’s protocol respectively.

Techniques: Migration, Control, Standard Deviation, Concentration Assay, Expressing

Journal: Cell reports

Article Title: PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma

doi: 10.1016/j.celrep.2023.112679

Figure Lengend Snippet: (A) Confocal microscopic image from “sandwich cultures” shows layered cells expressing either GPR133 (green) or PTK7 (red). Top view from a central confocal slice (center panel: x and y dimension) and orthogonal views (upper panel: z and x; right panel: z and y) depicted on the same scale. Scale bars, 10 μM. (B) HTRF assays in “sandwich cultures” demonstrate that PTK7 in neighboring cells increased cAMP levels in cells expressing wild-type (WT) cleaved GPR133, but not the uncleavable H543R mutant. A secreted PTK7 with no transmembrane domain (no TM, aa 1–703) did not influence GPR133 signaling. Two-way ANOVA: middle layer F 2, 96 = 4.62, not significant; outer layer F 2, 96 = 3.93, not significant; interaction of inner and outer layer F 4, 96 = 7.29, p < 0.0001. Tukey’s multiple comparisons: GPR133-WT-expressing cells with empty vector vs. PTK7 co-culture, p < 0.0001; GPR133-WT-expressing cells with full-length PTK7 vs. secreted PTK7 (no TM) co-culture, p < 0.0001. All other comparisons not significant; n = 4–22 independent experiments. (C) Pre-coating wells with PTK7 NTF (aa 1–703) significantly increased cAMP levels in WT GPR133-expressing, but not H543R uncleavable mutant-expressing cells. COL6 (native purified human collagen VI) had no effect on signaling. Two-way ANOVA: GPR133 variant expressed F 2, 80 = 11.65, p < 0.0001; protein coating F 2, 80 = 12.90, p < 0.0001; interaction of factors F 4, 80 = 9.24, p < 0.0001. Tukey’s multiple comparisons: GPR133-WT-expressing cells on PTK7 NTF-coated vs. uncoated dishes, p < 0.0001; GPR133-WT-expressing cells on PTK7 NTF-coated vs. COL6-coated dishes, p < 0.0001). All other comparisons not significant; n = 5–15 independent experiments. (D) Combinatorial effect of PTK7 binding and p13 Stachel peptide treatment on GPR133 signaling. HEK293T cells expressing either WT GPR133 or empty vector control were seeded onto PTK7 NTF-coated (1.17 μg/cm 2 ) or control-coated wells. Cells were then treated with synthetic p13 Stachel peptide (500 μM), inactive control peptide (500 μM), or solvent controls, and cAMP levels were measured by HTRF. The combination of PTK7 NTF binding and Stachel peptide elicited an additive response in GPR133 signaling compared with the individual treatments. Two-way ANOVA, GPR133 expression effect F 1, 48 = 365.1, p < 0.0001; treatment effect F 7, 48 = 13.2, p < 0.0001; interaction of GPR133 expression and treatment F 7, 48 = 13.2, p < 0.0001. Tukey’s multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 4 independent experiments. Data are depicted as mean ± SEM. See also and .

Article Snippet: Human Collagen Type VI , Rockland , 009-001-108.

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Co-Culture Assay, Purification, Variant Assay, Binding Assay, Control, Solvent

Journal:

Article Title: A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis

doi: 10.1172/JCI200318038

Figure Lengend Snippet: Immunofluorescent staining score for ECM proteins at d6. Glomerular staining for FN-EDA+ (a), laminin (b), type I collagen (c), and type III collagen (d) were lower in the PAI-1R–treated, nephritic group. *P < 0.001 compared with normal control. #P < 0.01 compared with disease control.

Article Snippet: Monoclonal mouse anti-cellular fibronectin extra domain positive (FN-EDA+) (Harlan Sera-Lab Ltd., Loughborough, United Kingdom), rabbit anti–mouse laminin (ICN Immunobiologicals, Aurora, Ohio, USA), goat anti–human type I collagen, and goat anti–human type III collagen (Southern Biotechnology Associates, Birmingham, Alabama, USA) were used as the primary Ab’s for detection of matrix components in glomeruli at d6.

Techniques: Staining

Journal:

Article Title: A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis

doi: 10.1172/JCI200318038

Figure Lengend Snippet: Relative glomerular mRNA expression of TGF-β1, PAI-1, fibronectin, and type I collagen. Glomerular mRNA levels of fibronectin and type I collagen were significantly reduced at d6, but the overexpression of TGF-β1 and PAI-1 was not changed in the PAI-1R–treated group. *P < 0.001 vs. normal control; #P < 0.02 vs. disease control; ‡P < 0.01 vs. disease control.

Article Snippet: Monoclonal mouse anti-cellular fibronectin extra domain positive (FN-EDA+) (Harlan Sera-Lab Ltd., Loughborough, United Kingdom), rabbit anti–mouse laminin (ICN Immunobiologicals, Aurora, Ohio, USA), goat anti–human type I collagen, and goat anti–human type III collagen (Southern Biotechnology Associates, Birmingham, Alabama, USA) were used as the primary Ab’s for detection of matrix components in glomeruli at d6.

Techniques: Expressing, Over Expression