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: Activation of the AMPK signalling pathway on the differentiation of hiPSCs to SANLCs. ( A ) Schematic diagram of the experimental protocol. ( B ) Different concentrations of an AMPK AA were added on Day 4 of differentiation for 2 days, and the expression levels of SAN cell markers SHOX2 , TBX18 , TBX3 , HCN4 , and pan-cardiomyocyte marker TNNT2 were analysed by qRT–PCR on Day 21 of differentiation. ( C ) AA was added on Days 4–6, 6–8, and 4–8, respectively, and the expression levels of SAN cell markers SHOX2 , TBX18 , TBX3 , and HCN4 and pan-cardiomyocyte marker TNNT2 were analysed by qRT–PCR on Day 21 of differentiation. One-way ANOVA followed by Bonferroni post hoc test was performed (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; 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: Membranes were blocked in 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-SHOX2 antibody (1:1000) (ab55740, Abcam, USA), anti-HCN4 antibody (1:1000) (MA3-903, Invitrogen, USA), anti-NKX2.5 antibody (1:1000) (ab91196, abcam, USA), and anti-HSP90 antibody (sc-13119, Santa Cruz Biotechnology, USA).

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

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: Inhibition of the AMPK signalling pathway on the differentiation of hiPSCs to SANLCs. ( A ) Schematic diagram of the experimental protocol. ( B ) Different concentrations of an AMPK inhibitor CC were added on Day 6 of differentiation induction for 2 days, and the expression levels of SAN cell markers SHOX2 , TBX18 , TBX3 , and HCN4 and pan-cardiomyocyte marker TNNT2 were analysed by qRT–PCR on Day 21 of differentiation. ( C ) AA was added on Days 6–8, 8–10, and 6–10, and the expression levels of SHOX2, TBX18, TBX3 , HCN4 , and TNNT2 were analysed by qRT–PCR on Day 21 of differentiation. One-way ANOVA followed by Bonferroni post hoc test was performed (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; 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: Membranes were blocked in 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-SHOX2 antibody (1:1000) (ab55740, Abcam, USA), anti-HCN4 antibody (1:1000) (MA3-903, Invitrogen, USA), anti-NKX2.5 antibody (1:1000) (ab91196, abcam, USA), and anti-HSP90 antibody (sc-13119, Santa Cruz Biotechnology, USA).

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

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 effect of the combined protocol (AC group) on the differentiation of hiPSCs into SANLCs. ( A ) Schematic diagram of the experimental protocol; ( B ) 800 μM AA was added on Days 4–6 of differentiation, and 10 μM CC was added on Days 6–8, and then the expression levels of SHOX2 , TBX18 , TBX3 , HCN4 , and TNNT2 were analysed by qRT–PCR on Day 21 of differentiation. One-way ANOVA followed by Bonferroni post hoc test was performed (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, N = 5). All PCR expression values were normalized to the housekeeping gene GAPDH. Data are presented as ‘mean ± SEM’.

Article Snippet: Membranes were blocked in 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-SHOX2 antibody (1:1000) (ab55740, Abcam, USA), anti-HCN4 antibody (1:1000) (MA3-903, Invitrogen, USA), anti-NKX2.5 antibody (1:1000) (ab91196, abcam, USA), and anti-HSP90 antibody (sc-13119, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Quantitative RT-PCR

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: Protein-level validation of SANLC differentiation following the AC protocol. ( A–C ) Immunofluorescence staining for cTNT co-stained with HCN4 ( A ), SHOX2 ( B ), or NKX2.5 ( C ). Nuclei were counterstained with DAPI. Images were acquired using a Leica SP8 confocal microscope with a 40× immersion objective. Scale bars = 50 μm. ( D–F ) Western blot analysis of HCN4 ( D ), SHOX2 ( E ), and NKX2.5 ( F ) expression in control and AC groups. HSP90 was used as a loading control. Bar graphs show relative protein levels. N = 3 biological replicates. ( G ) Representative flow cytometry plots showing co-staining of cTNT and NKX2.5 in control and AC groups. cTNT⁺/NKX2.5⁻ cells are defined as SANLCs. ( H ) Quantification of SANLCs (cTNT⁺/NKX2.5⁻) from flow cytometry analysis ( N = 4 biological replicates). Data are shown as mean ± SEM. Statistical significance: ** P < 0.01, **** P < 0.0001 (unpaired t -test).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-SHOX2 antibody (1:1000) (ab55740, Abcam, USA), anti-HCN4 antibody (1:1000) (MA3-903, Invitrogen, USA), anti-NKX2.5 antibody (1:1000) (ab91196, abcam, USA), and anti-HSP90 antibody (sc-13119, Santa Cruz Biotechnology, USA).

Techniques: Biomarker Discovery, Immunofluorescence, Staining, Microscopy, Western Blot, Expressing, Control, Flow Cytometry