Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Adipose Mesenchymal Cells-Derived EVs Alleviate DOCA-Salt-Induced Hypertension by Promoting Cardio-Renal Protection

doi: 10.1016/j.omtm.2019.11.002

Figure Lengend Snippet: ASC-EV Treatment Promoted Important Attenuation of Kidney Fibrosis in the DOCA-Salt Model (A) Representative photomicrographs of kidney slices stained with Picrosirius red on 9-week-treated rats from the 4 experimental groups (scale bars, 50 μm). (B) α-SMA expression in the kidney of 9-week-treated rats under the different experimental conditions was determined by immunohistochemistry (n = 4 for each treatment) (scale bars, 50 μm). (C) Quantification of Picrosirius red stained area in kidney sections expressed as a percentage of the total area of the kidney, in 9-week-treated rats, measured for all the animals tested in the 4 experimental groups (SHAM, n = 4; UNX, n = 9; DOCA, n = 13; DOCA-EV, n = 10). (D−F) Western blot analysis of α-SMA and Desmin in the cortex of 9-week-treated UNX (n = 6), DOCA, and DOCA-EV (n = 8) rats. GAPDH was used as loading control. Values represent the fold increase with respect to UNX (considered as the reference = 1). (G−I) Analysis of COL1A1 , COL4A1 , and FN mRNA levels in the renal tissue in control SHAM (n = 3) and in all the uninephrectomized animal groups (UNX, n = 6; DOCA and DOCA-EV, n = 7). qRT-PCR data were expressed as RQ and UNX was used as the reference sample. Data represent mean ± SE. *indicates statistical difference to SHAM group, # indicates statistical difference to UNX group, and § indicates statistical difference to DOCA group; p < 0.05.

Article Snippet: The following primary antibodies were used: GAPDH, sc-32233 (1:1,000, Santa Cruz); α-SMA, MAB1420 (1:1,000, R&D Systems, Minneapolis, MN, USA); Desmin, ab32362 (1:1,000, Abcam); B cell lymphoma 2 (Bcl2), 2876 (1:1,000, Cell Signaling Technology, Danvers, MA, USA); and Bcl-2-associated X (Bax), 2772S (1:500, Cell Signaling Technology).

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Control, Quantitative RT-PCR

Journal: Annals of Gastroenterological Surgery

Article Title: Intratumoral Fusobacterium nucleatum Drives Cancer‐Associated Fibroblasts Enrichment and Immune Exclusion in Esophageal Squamous Cell Carcinoma

doi: 10.1002/ags3.70116

Figure Lengend Snippet: Histopathological assessment of CAFs in ESCC. (a) Representative FISH images of ESCC tissues showing signals for F. nucleatum (red; FUS664), all bacteria (green; EUB338), and nuclei (blue; DAPI). Positive F. nucleatum signals were observed in cases that were identified as F. nucleatum –positive by qPCR. All images were acquired at ×200 magnification. (b) Representative FISH images of ESCC tissues obtained by laser scanning confocal microscopy, demonstrating intracellular signals of Fusobacterium nucleatum (red; FUS664) within tumor cells. Nuclei were counterstained with DAPI (blue). Images were acquired at ×400 magnification. (c) Representative FISH images of ESCC tissues showing signals for F. nucleatum (red; FUS664), all bacteria (green; EUB338), and nuclei (blue; DAPI). F. nucleatum signals were observed within tumor cells located in regions enriched with α‐SMA–positive CAFs. The boxed areas indicate regions of interest, imaged at ×200 magnification. ** p < 0.01.

Article Snippet: We used monoclonal mouse anti α‐SMA antibody (1:250; #56856; Cell Signal Technology) and RelA (1:800; #8242; Cell Signal Technology) as the primary antibody, following the manufacturer's protocols.

Techniques: Bacteria, Confocal Microscopy

Journal: Annals of Gastroenterological Surgery

Article Title: Intratumoral Fusobacterium nucleatum Drives Cancer‐Associated Fibroblasts Enrichment and Immune Exclusion in Esophageal Squamous Cell Carcinoma

doi: 10.1002/ags3.70116

Figure Lengend Snippet: Immunohistochemical analysis of NF‐κB activation and its association with F. nucleatum and CAFs in ESCC. (a) The proportion of NF‐κB–positive tumors was significantly higher in F. nucleatum –positive cases than in F. nucleatum –negative cases. (b) Representative immunohistochemical staining on adjacent serial sections of ESCC tissues showing stromal α‐SMA expression and nuclear RelA localization in tumor cells. These signals were observed in close proximity. Images were acquired at ×200 magnification. (c) Dual positivity for stromal α‐SMA and NF‐κB–positive in tumor cells was significantly enriched in F. nucleatum –positive tumors compared to F. nucleatum –negative tumors. (d) Summary of the results. F. nucleatum contributes to the progression of ESCC by inducing NF‐κB–mediated inflammatory signaling in tumor cells and promoting the activation of CAFs. ** p < 0.01.

Article Snippet: We used monoclonal mouse anti α‐SMA antibody (1:250; #56856; Cell Signal Technology) and RelA (1:800; #8242; Cell Signal Technology) as the primary antibody, following the manufacturer's protocols.

Techniques: Immunohistochemical staining, Activation Assay, Staining, Expressing

Journal: Biomaterials

Article Title: Fibrin polymer on the surface of biomaterial implants drives the foreign body reaction.

doi: 10.1016/j.biomaterials.2021.121087

Figure Lengend Snippet: Fig. 9. Macrophages within the fibrous capsule express α-SMA and CD68. PCTFE sections were implanted in WT (A, B) and FibAEK (C, D) mice for 3, 7, and 14 days. Representative images of the capsule sam ples retrieved from WT (n = 3) and FibAEK

Article Snippet: The mouse anti-α-SMA mAb (catalog number MAB1420-SP) was from R&D systems.

Techniques:

Journal: Biomaterials

Article Title: Fibrin polymer on the surface of biomaterial implants drives the foreign body reaction.

doi: 10.1016/j.biomaterials.2021.121087

Figure Lengend Snippet: Fig. 10. Macrophages within the fibrous capsule express collagen. PCTFE sections were implanted in WT and FibAEK mice for 14 days and the cells accumulated within the capsules were isolated as described in the Materials and Methods. (A) The cells were allowed to adhere to the surface of a FluoroDish, fixed, and incubated with Alexa Fluor 568-conjugated phalloidin, anti-α-SMA, and anti-CD68 antibodies. Representative confocal images of myofibroblasts and macrophages isolated from the capsule retrieved from WT mice are shown. Arrowheads point to podosomes seen in macrophages incubated with phalloidin-Alexa Fluor 568. The scale bar is 10 μm. (B) Quantification of cells expressing α-SMA only (myofibroblasts) or both α-SMA and CD68 (macrophages). Approximately 2000–2500 cells isolated from the capsules retrieved from each WT and FibAEK mice were analyzed with ~400 cells present in each field of view. (C) The cells isolated from the capsule obtained from WT mice were incubated with anti-CD11b mAb M1/70 and anti-collagen I mAb. Myofibroblasts shown in the upper panel are indicated by white arrows. The scale bar is 20 μm. (D) Representative confocal images of collagen-expressing macrophages isolated from the WT and FibAEK mice. The cells were labeled for collagen 1 (green) and actin (red). The scale bar is 10 μm. (E) The frequency distribution of fluorescence intensities for collagen I in macrophages isolated from the capsules retrieved from WT and FibAEK mice was expressed as CTCF arbitrary units (A.U). A total of 300 cells from 5 random fields in the samples prepared from WT and FgAEK

Article Snippet: The mouse anti-α-SMA mAb (catalog number MAB1420-SP) was from R&D systems.

Techniques: Capsules, Isolation, Incubation, Expressing, Labeling, Fluorescence

Journal: Nature communications

Article Title: Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome.

doi: 10.1038/s41467-023-36818-0

Figure Lengend Snippet: Fig. 2 | UBE3A knockdown increases cofilin and decreases F-actin content and PIEZO2 function. a Top, representative whole-cell patch-clamp recordings of currents elicited by mechanical stimulation (−60 mV) in MCC13 cells transfected with scrambled, UBE3A, or PIEZO2 siRNAs. Bottom, current densities elicited by maximum displacement of siRNA-transfected cells. Bars are mean ± SD. Kruskal- Wallis (H = 18.76; p = 8.4−5) and Dunn’s multiple comparisons test. b Top, western blot (anti-PIEZO2) of the membrane fractions of MCC13 cells transfected as in (a). Bottom, mean/scatter-dot plot showing relative intensities of PIEZO2 protein nor- malized to PIEZO2 in the Sc. group. Lines are mean ± SD. Kruskal-Wallis (H = 12.78; p = 0.0017) and Dunn’s multiple comparisons test. c Top, currents elicited by mechanical stimulation (−60 mV) in cells transfected with UBE3A plasmid. Bottom, current densities elicited by maximum displacement of UBE3A transfected cells. Bars are mean ± SD. Two-tailed unpaired t-test with Welch’s correction (t = 3.9). d Top, western blot (anti-PIEZO2) of the membrane fractions of MCC13 cells transfected with UBE3A plasmid. Bottom, mean/scatter-dot plot showing relative intensities of PIEZO2 protein in UBE3A transfected cells normalized to PIEZO2 in the control group. Lines are mean ± SD. Two-tailed one-sample t-test (t = 4.4). e Top, currents elicited by mechanical stimulation (−60 mV) of latrunculin A (1 µM; 24 h)- treated MCC13 cells. Bottom, current densities elicited by maximum displacement. Bars are mean ± SD. Two-tailed unpaired t-test with Welch’s correction (t = 9.9). f Top, western blot (anti-PIEZO2) of the membrane fractions of MCC13 cells treated

Article Snippet: Protein concentrations were measured with the Bio-Rad protein assay or the PierceTM BCA Protein Assay Kit (ThermoFisher Scientific), and equivalent protein amounts were loaded in Mini-PROTEAN TGX Stain-Free Precast Gels (Bio-Rad).Mousemonoclonal anti-UBE3A (1:1,000; Sigma-AldrichCat# SAB1404508, RRID:AB_10740376), mouse monoclonal anti-actin for MCC13 cells (1:5,000; Bio-Rad Cat# MCA358GT, RRID:AB_323521), mouse monoclonal anti-actin for DRGs (1:1,000; Cytoskeleton Cat# AAN02, RRID:AB_2884962), goat polyclonal anti-mouse IgGH&L (HRP) (1:20,000; Abcam Cat# ab205719, RRID:AB_2755049), rabbit polyclonal anti-human PIEZO2 (1:1,000; abcepta Cat# AP16313b, RRID:AB_11136435), rabbit polyclonal anti-cofilin (1:1,000; abcepta Cat# AP53892, RRID:AB_2923306), rabbit monoclonal anti-cofilin for DRGs (1:1,000; Cell Signaling Technology Cat# 5175, RRID:AB_10622000), and goat anti-rabbit IgG HL-HRP conjugated (1:10,000; Bio-Rad Cat# 1706515, RRID:AB_2617112) antibodies were used for western blots.

Techniques: Knockdown, Patch Clamp, Transfection, Western Blot, Membrane, Plasmid Preparation, Two Tailed Test, Control