METTL3 affected calcium transients in atrial cardiomyocytes via PCDHGA10. (A) Calcium transients were detected by the confocal microscopy in PCDHGA10-mcherry and mcherry (control) NMAMs. Data are represented as mean ± SD. mcherry (n = 14 cells from three technical replicates), PCDHGA10-mcherry (n = 18 cells from three technical replicates). ***P < 0.001, comparisons made using unpaired Student’s t-test. (B) Calcium transients were detected by the confocal microscopy in sh-Ctrl, sh-Mettl3, sh-Pcdhga10, and sh-Mettl3 + sh-Pcdhga10 NMAMs. Data are represented as mean ± SD. Sh-Ctrl (n = 32 cells from three technical replicates), sh-Mettl3 (n = 39 cells from three technical replicates), sh-Pcdhga10 (n = 37 cells from three technical replicates), and sh-Mettl3 + sh-Pcdhga10 (n = 41 cells from three technical replicates). *P < 0.05, ****P < 0.0001, comparisons made using two-way ANOVA. (C) Immunofluorescence staining with SERCA2a antibody was performed in NMAMs overexpressing PCDHGA10-GFP. The sarcoplasmic reticulum was demonstrated by the ER tracker-red. The white arrows show the sites where PCDHGA10 colocalizes with SERCA2a and the sarcoplasmic reticulum. (D) Co-IP experiment of HEK293T cells overexpressing PCDHGA10-GFP and Flag-SERCA2a was performed with GFP and Flag antibodies, followed by western blotting experiments with GFP and Flag antibodies. (E) SERCA2a-ATPase activity measurement in HL-1 cells overexpressing PCDHGA10-GFP and GFP (Control). Data are represented as mean ± SD. GFP (n = 4), PCDHGA10-GFP (n = 4), comparison made using unpaired Student’s t-test. (F) SERCA2a ATPase activity measurement in sh-Ctrl, sh-Mettl3, sh-Pcdhga10, and sh-Mettl3 + sh-Pcdhga10 HL-1 cells. Data are represented as mean ± SD. Sh-Ctrl (n = 3), sh-Mettl3 (n = 3), sh-Pcdhga10 (n = 3), and sh-Mettl3 + sh-Pcdhga10 (n = 3). *P < 0.05, **P < 0.01, comparisons made using two-way ANOVA. ANOVA, analysis of variance; GFP, green fluorescent protein; METTL3, methyltransferase like 3; NMAMs, neonatal mice atrial myocytes; PCDHGA10, protocadherin gamma subfamily A, 10; SERCA2a, SR Ca2+-ATPase Type 2a; SD, standard deviation.
