Journal: Europace

Article Title: Promoting differentiation of human-induced pluripotent stem cells into sinoatrial node-like cells through programmed regulation of AMPK signalling pathway

doi: 10.1093/europace/euaf288

Figure Lengend Snippet: The role of AMPK signalling pathway in NKX2.5 expression. ( A ) Pan-cardiomyocyte differentiation protocol. CHIR (Wnt agonist) and IWP2 (Wnt inhibitor) were used for directed differentiation of cardiac cells. ( B–D ) Expression trends of markers at various stages during cardiomyocyte differentiation including cardiac mesoderm marker MESP1, cardiomyocyte progenitor marker NKX2.5, and cardiomyocyte marker TNNT2. qRT–PCR analysis was conducted to observe the dynamic expression trends from Days 0 to 13 of induced differentiation. ( E–F ) KEGG analysis of signalling pathways enriched with NXK2.5 expression. ( G ) Different concentrations of the AMPK agonist AICAR (AA) were added on Day 4 of differentiation for 2 days. The expression level of NKX2.5 in each group was analysed by qRT–PCR on Day 6. ( H ) Different concentrations of AMPK inhibitor CC were added on Day 4 of differentiation for 2 days. The expression level of NKX2.5 in each group was analysed by qRT–PCR on Day 6. One-way ANOVA followed by Bonferroni post hoc test was performed, * P < 0.05, *** P < 0.001, **** P < 0.0001, and ns, not significant vs. control, N = 5. All PCR expression values were normalized to the housekeeping gene GAPDH. Data are presented as ‘mean ± SEM’.

Article Snippet: At Day 0, the medium was replaced with RPMI/B-27 containing 8 μM CHIR99021 (a GSK3 inhibitor) (S1263, Selleck, USA) without insulin and incubated for 48 h. Subsequently, the medium was refreshed with RPMI/B-27 containing 5 μM IWP2 (a Wnt inhibitor) (S7085, Selleck, USA) without insulin for 48 h. On Days 4–6, the medium was switched to RPMI/B-27 without insulin.

Techniques: Expressing, Marker, Quantitative RT-PCR, Control

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