Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules

doi: 10.1007/7651_2021_394

Figure Lengend Snippet:

Article Snippet: Endo-IWR 1 , MedChemExpress , HY-12238 , WNT pathway inhibitor.

Techniques: Positive Control

Journal: Frontiers in Oncology

Article Title: AXL Overexpression in Tumor-Derived Endothelial Cells Promotes Vessel Metastasis in Patients With Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.650963

Figure Lengend Snippet: AXL/SOX2/DKK-1 axis in HUVECs promotes HCC metastasis. (A) DKK-1 and CCL14 secretion was significantly downregulated in CM from HUVEC-AXL-KD compared with that from HUVEC-AXL-NC, as detected with a human cytokine antibody array. (B) DKK-1 and CCL14 expression was markedly upregulated in the CM of HUVECs overexpressing AXL (CCL14: p < 0.001; DKK-1: p = 0.003) compared with the CM of HUVEC-AXL-NC, as detected by ELISA assay. (C) AXL siRNA downregulated DKK-1 and CCL14 secretion in the CM of HUVEC-AXL-NC and HUVEC-AXL-OE cells (CCL14: p < 0.001 and p < 0.001; DKK-1: p < 0.001 and p < 0.001). (D) DKK1 siRNA (MHCC-97L: p < 0.001 and p < 0.001; HCC-LM3: p <0.001 and p < 0.001), but not CCL14 siRNA (MHCC-97L: p = 0.126 and p = 0.711; HCC-LM3: p = 0.901 and p = 0.694) could attenuate the effect of the CM from HUVEC-AXL-NC and HUVEC-AXL-OE cells on the migration of HCC-LM3 cells and MHCC-97L cells. (E) SOX2 mRNA expression was significantly increased in HUVEC-AXL-OE cells and decreased in HUVEC-AXL-KD cells compared with HUVEC-AXL-NC cells (HUVEC-AXL-KD: p < 0.001, HUVEC-AXL-OE: p < 0.001). (F) AXL overexpression could significantly increase SOX2 and DKK-1 protein expression in HUVEC-AXL-OE cells compared with HUVEC-AXL-NC cells, and SOX2 siRNA inhibited SOX2 and DKK-1 protein expression in HUVEC-AXL-OE and HUVEC-AXL-NC cells.

Article Snippet: The protein concentrations of CCL14 and DKK-1 in the supernatants were also measured using an enzyme-linked immunosorbent assay (ELISA) kit (CCL14: EK1123 Boster, Wuhan, China; DKK-1: EK0867 Boster, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Ab Array, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression

Journal: STAR Protocols

Article Title: Protocol for serial organoid formation assay using primary colorectal cancer tissues to evaluate cancer stem cell activity

doi: 10.1016/j.xpro.2022.101218

Figure Lengend Snippet: Examples of organoid detection methods using high content imaging (A) 3D-embedded organoids can be detected, counted, and scored by high content imaging using ImageXpress Automated Cell Imaging Systems, or equivalent platforms. (B) Example of organoid detection based on a size exclusion threshold, where cellular structures below a definite size are not counted from brightfield images. A color mask is applied by the analysis software over each structure considered as an organoid. Scale bar = 70 μm. (C) Example for the use of a fluorescent (GFP) reporter system outlining a specific population of HCT116 organoids to be counted in an experiment. A color mask is applied by the analysis software over each structure presenting a fluorescence signal above a definite background threshold (FLUOR Mask). Scale bar = 70 μm. (D) Identification of live organoid structures using the cell permeable, fluorescent dye Calcein AM (green). While several live organoids are detectable in DMSO-treated wells, only residual live cells are observed upon treatments with the anticancer small molecule YB-0158. Scale bar = 70 μm.

Article Snippet: YB-0158 , MedChemExpress , Cat# HY-136541.

Techniques: Imaging, Software, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for serial organoid formation assay using primary colorectal cancer tissues to evaluate cancer stem cell activity

doi: 10.1016/j.xpro.2022.101218

Figure Lengend Snippet: Schematic analysis of the colorectal tumor organoids resulting from serial passaging (A) Brightfield images of secondary organoids depicting size difference over the growth stages. Scale bar = 70 μm. (B) High content imaging can be used to establish organoid counts, size, and shape (form factor: FF) variations for control vs. treated conditions. (C) Secondary organoid counts obtained from the serial passage of primary organoid cultures treated with 3 compounds targeting CSC functions in colorectal tumors (BIX01294, UNC0642: G9a inhibitor. YB-0158: Sam68 modulator). Organoid counts were established based on size exclusion (< 40 μm). n≥4 biological replicates from 3 independent patients; ∗∗: p≤0.0053, ∗∗∗: p≤0.0001). (D) Fluorescence imaging of a colorectal tumor organoid by confocal microscopy. A composite image of proliferative cells (Ki-67; green), adherens junctions (E-Cadherin; red), and nuclei (Hoechst 33342; blue) is presented. Scale bar = 30 μm. (E) Tridimensional reconstruction of CRC patient-derived spheroids and organoids from confocal fluorescence microscopy images using the Imaris 9.5 rendering platform. For each type of multicellular structure, immunostaining of the proliferation marker Ki-67 and intestinal self-renewal marker Bmi1 are presented. Scale bar = 30 μm.

Article Snippet: YB-0158 , MedChemExpress , Cat# HY-136541.

Techniques: Passaging, Imaging, Fluorescence, Confocal Microscopy, Derivative Assay, Microscopy, Immunostaining, Marker

Journal: Heliyon

Article Title: Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo

doi: 10.1016/j.heliyon.2024.e28021

Figure Lengend Snippet: Cap inhibits Pi-induced calcification of VSMCs by inhibiting the Wnt/β-catenin signaling pathway by upregulation of TRPV1 receptor expression. A) RT-qPCR showing the mRNA levels of Wnt3a and β-catenin; B) Western blot illustrating the protein expression levels of Wnt3a and β-catenin; C) Alizarin Red staining assay; D) Calcium content in VSMCs assessed using a calcium colorimetric assay kit. E) RT-qPCR showing the expression of osteogenesis-specific and phenotypic markers in VSMC calcification. F) Western blot depicting the protein expression levels of osteogenesis-specific and phenotypic markers in VSMC calcification; G) Western blot showing the protein expression of β-catenin after treatment with DKK1. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively; Pi + Cap: VSMCs cultured in the pro-calcifying medium with 20 μM Cap; Pi + Cap + CPZ: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 10 μM CPZ; Pi + Cap + DKK1: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 20 ng/mL DKK1; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ※: p < 0.05, ※※: p < 0.01, ※※※: p < 0.001 vs Pi + Cap; ns: p > 0.05 vs Control; p > 0.05 vs Pi; p > 0.05 vs Pi + Cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies against BMP-2 (PRP100126) and Runx2 (ABP53087) were purchased from Abbkine (Wuhan, Hubei, China); SM22α (AF9266) and Wnt3a (DF6113), Affinity Biosciences LTD; β-actin (81115-1-RR), proteintech (Hubei, China); β-catenin (ab32572), Abcom (USA); and DKK1 ( HY-P73305 ), MCE China (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Calcium Colorimetric Assay, Cell Culture, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Aged garlic extract enhances the production of β‑defensin 4 via activation of the Wnt/β‑catenin pathway in mouse gingiva

doi: 10.3892/etm.2024.12791

Figure Lengend Snippet: Effect of AGE on the β-defensin 4 production and canonical Wnt pathway in GE1 mouse gingival epithelial cells. (A) Cells were treated with AGE at the indicated concentrations (0.25-2 mg/ml) for 24 h. The amount of β-defensin 4 protein secreted into the culture medium was determined using an ELISA. The graph shows the concentration of β-defensin 4 in the medium. Data are presented as the mean ± standard deviation (n=6). * P<0.05 (Dunnett's test). (B) Cells were treated with AGE (2 mg/ml) for the indicated duration (1-24 h). The protein level of β-catenin in the nucleus was analyzed by western blotting. Images show representative data of western blotting for β-catenin with lamin B1 as the internal control. The graph shows the level of β-catenin protein normalized to that of lamin B1. Data are presented as the mean ± standard deviation (n=3-7). * P<0.05 (Mann-Whitney U test). (C) Cells were treated with AGE for 24 h in the presence or absence of LF3 (30 µM; 30-min pretreatment), an inhibitor of β-catenin in the canonical Wnt pathway. The amount of β-defensin 4 protein secreted into the culture medium was determined using an ELISA. The graph shows the concentration of β-defensin 4 in the medium. Data are presented as the mean ± standard deviation (n=4). * P<0.05, ** P<0.01 (Holm-Bonferroni test). A, AGE; AGE, aged garlic extract; C, control.

Article Snippet: A canonical Wnt signaling pathway specific inhibitor LF3 and a glycogen synthase kinase-3 (GSK-3) specific inhibitor 6-bromoindirubin-3'-oxime (BIO) were from Cayman Chemical (Ann Arbor).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Western Blot, Control, MANN-WHITNEY