Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 6. Effects of Inhibiting BMP, Canonical Wnt, and Nodal signaling on Germ Lineage Differentiation. A-B) Representative images of Brachyury (green), Otx2 (red), and DAPI (blue) immunostaining of WT and Dvl TKO EBs on day 7 of differentiation after inhibition with BMP (LDN-193189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB-431542) drug treatments. Scale bar ¼ 100 μm. C-D) Quantification of Brachyury and Otx2-positive cells in WT and Dvl EBs differentiated for 5–7 days (n ¼ 20/group). Brachyury-positive cells are not detectable in Dvl TKO EBs, and there are no significant differences in Otx2-positive cells. E-F) Quantitative RT-PCR analysis of Brachyury (mesoderm) and GATA4 (endoderm) mRNA expression to determine differences in differentiation potential in WT and Dvl TKO EBs over a 6-day time course (n ¼ 3). Expression is normalized to the untreated controls in Fig. 5G (Brachyury) and 5H (GATA4). E) Brachyury expression in WT EBs is attenuated with inhibition of BMP, Wnt, and Nodal signaling, similar to Dvl TKO EBs. F) Inhibition of BMP and Wnt signaling significantly enhances endoderm differentiation in WT and Dvl TKO EBs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Immunostaining, Inhibition, Quantitative RT-PCR, Expressing

Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 7. Effects of Developmental Pathway Modulation on Germ Lineage Differentiation. Activation of BMP (BMP4), canonical Wnt (Wnt3a and CHIR-99021) and Nodal (Activin A) pathways in WT EBs enhances mesoderm (Bra) differentiation, while endoderm (GATA4) differentiation remains low, and ectoderm (Otx2) dif- ferentiation diminishes over time. Treatment with these activators in Dvl TKO EBs does not rescue mesoderm induction. The relative level of endoderm marker expression is higher in Dvl TKO EBs compared to WT EBs treated with Wnt activators, Wnt3a and CHIR, but not BMP4 or Activin A. Dvl loss also delays the decrease in ectoderm lineage expression. Inhibition of BMP (LDN-198189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB431542) pathways in WT EBs results in the suppression of mesoderm differentiation, recapitulating the Dvl TKO phenotype. Pathway inhibitor treatment in WT EBs, leads to similar trends of differentiation as Dvl TKO EBs. Endoderm differentiation is greatly enhanced in Dvl TKO EBs with LDN-193189 and IWP L6 treatment.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Activation Assay, Marker, Expressing, Inhibition

Journal: Current Research in Microbial Sciences

Article Title: The lncRNA GAS5-encoded micropeptide facilitates influenza virus replication through modulation of the Wnt/β-catenin signaling pathway

doi: 10.1016/j.crmicr.2026.100559

Figure Lengend Snippet: GAS5-P50 interacts with NOTUM to enhance Wnt/β-catenin activation. (A) Proteins interacting with GAS5-P50 were identified through immunoprecipitation (IP) and subsequent silver staining. (B) Molecular docking of GAS5-P50 and NOTUM protein. (C, D) 293T cells were co-transfected with GAS5-P50-Flag and NOTUM-HA plasmids, followed by infection with PR8 influenza virus. GAS5-P50-Flag was immunoprecipitated from the lysates with anti-Flag antibody and the precipitates were blotted with anti-HA antibody (C). NOTUM-HA was immunoprecipitated from the lysates with anti-HA antibody and the precipitates were blotted with anti-Flag antibody (D). (E) 293T cells were transfected with either EV or GAS5-P50-Flag plasmids, followed by infection with PR8 influenza virus. GAS5-P50-Flag was immunoprecipitated from the lysates with anti-Flag antibody and the precipitates were subjected to immunoblotting with anti-NOTUM antibody. (F) 293T cells were transfected with EV, GAS5-P50, NOTUM, or GAS5-P50 together with NOTUM, respectively. After 16 h of infection with PR8 influenza virus, viral titers in the supernatants were determined by plaque assay. (G) 293T cells were transfected with EV, GAS5-P50, NOTUM, or GAS5-P50 together with NOTUM, followed by treatment with Wnt3a (1 μg/mL). The expression of β-catenin was examined by Western blotting. (H, I) 293T cells were transfected with EV, GAS5-P50, NOTUM, or GAS5-P50 together with NOTUM, respectively. After 16 h of infection with PR8 influenza virus, the expression of AXIN2 (H) and LEF1 (I) was examined by RT-qPCR. (J, K) A549 cells were treated with Wnt3a (1 μg/mL) (J) or IWP-2 (50 μM) (K), followed by infection with PR8 influenza virus for 16 h. Viral titers in the supernatants were determined by plaque assay. Data are represented as mean ± SD; Shown are representative data from three biologically independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns represents no significance.

Article Snippet: Wnt3a and IWP-2 were purchased from MedChemExpress (New Jersey, USA).

Techniques: Activation Assay, Immunoprecipitation, Silver Staining, Transfection, Infection, Virus, Western Blot, Plaque Assay, Expressing, Quantitative RT-PCR