Journal: Frontiers in oncology

Article Title: Wnt/β-Catenin Inhibition Disrupts Carboplatin Resistance in Isogenic Models of Triple-Negative Breast Cancer.

doi: 10.3389/fonc.2021.705384

Figure Lengend Snippet: FIGURE 3 | b-catenin overexpression in 468’P induces carboplatin-resistance, pluripotency-related gene expression, and cancer stem cell features. (A) Western blot (top) of total b-catenin in MDA-MB-468 cells transduced with an empty vector or truncated, constitutively active b-catenin isoform DN90 and phase-contrast microscopy (down). (B) Enriched gene sets from Wikipathways database by one-tailed GSEA of ranked DEGs between 468’OE and 468’P sorted by normalized enrichment score (left) and enrichment map illustrating pathways most significantly different between 468’OE and 468’P (right). (C) Non-linear fit model of [CAR] vs. normalized response for IC50 determination. (468’OE: n=6, R2 = 0.92; 468’CTRL: n=6, R2 = 0.95). (D) Representative flow cytometry scatterplots of annexin V staining (left) of 468’CTRL and 468’OE cells treated with carboplatin 2 mM for 72h and statistical analysis of the mean frequency of annexin V positive cells using one-way ANOVA corrected for multiple comparisons using the Holm-Sidak method (right, n=3). (E) mRNA level fold change (Log2) of CTNNB1 (b-catenin), Wnt target AXIN2, and stem cell markers in 468’OE cells vs. 468’CTRL (n=4). Multiple t-tests with Holms-Sidak correction for multiple comparisons. (F) Representative scatterplots of flow cytometric analysis of aldehyde dehydrogenase activity (left) and statistical analysis of the mean percentage of ALDH+ cells in 468’OE (n=5) and 468’CTRL (n=5) using Welch’s t-test (right). (G) Representative scatterplots of flow cytometric analysis of CD44-PE and CD24-APC immunolabeling (left) and corresponding statistical analysis of the mean percentage of CD44+/CD24- cells using Welch’s t-test (right; n=3). (H) Representative brightfield images of tumorspheres generated from 468’CTRL and 468’OE cells (left, scale bar: 50 mm) and statistical analysis of mean tumorsphere forming units (number of spheres/number of seeded single cells) using Welch’s t-test (right; n=3). (Barplots represent mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non significant).

Article Snippet: For overexpression of DN90-b-catenin, we used pLV-betacatenin DN90 (Addgene, #36985) and pPRIME-CMV-NEOrecipient (CTRL, Addgene, #11659).

Techniques: Over Expression, Gene Expression, Western Blot, Transduction, Plasmid Preparation, Microscopy, One-tailed Test, Cytometry, Staining, Activity Assay, Immunolabeling, Generated

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Circulating Exosomal miR-20b-5p Inhibition Restores Wnt9b Signaling and Reverses Diabetes-Associated Impaired Wound Healing.

doi: 10.1002/smll.201904044

Figure Lengend Snippet: Figure 6. miR-20b-5p modulates Wnt9b/β-catenin Signaling to control angiogenesis. A) We sought to identify putative miR-20b-5p targets using miRbase, Targetscan, and miRanda, and 73 genes were identified as miR-20b-5p target gene. B) The association between miR-20b-5p and Wnt9b was demonstrated by luciferase reporter assay. Cells were cotransfected with these different constructs (10 µg) along with the appropriate miR-20b-5p mimic using Lipofectamine 3000, and a Dual-Luciferase Reporter Assay kit (Promega) was used to quantify luciferase activity after 48 h. SpectraMax M5 software (molecular devices, CA, USA) was used to analyze the resultant data. C) Effect of miR-20b-5p on the expression of Wnt9b assessed by western blotting. D) Western blotting analysis indicated that antagomiR-20b-5p could restore the levels of Wnt9b and β-catenin protein. E) EDU results

Article Snippet: Next, proteins were separated via 10% SDS-PAGE, transferred to PVDF membranes, and probed with the appropriate primary antibodies specific for CD81 (#ab219209, Abcam, Cambridge, UK, 1:1000), TSG101 (#ab30871, Abcam, Cambridge, UK, 1:1000), Cyclin D1 (#ab16663, Abcam, Cambridge, UK, 1:1000), Cyclin D3 (#ab28283, Abcam, Cambridge, UK, 1:1000), Bax (#2772, CST, MA, USA, 1:2000), and Bcl-2 (#ab185002, Abcam, Cambridge, UK, 1:2000), Wnt9b (#ab69287, Abcam, Cambridge, UK, 1:1000), β-catenin (#8480, CST, MA, USA, 1:2000).

Techniques: Control, Luciferase, Reporter Assay, Construct, Activity Assay, Software, Expressing, Western Blot