Fig 2: Knockdown of TRPC7 decreased the activity of RyR2 and SERCA without affecting the activity of IP3R in mESC-CMs. Representative CaTs recorded from the control group a before and b after applying ryanodine. Representative CaTs recorded from TRPC7 knockdown group c before and d after applying ryanodine. e, f Bar charts showing the normalized frequency and the Vmax-upstroke calculated from a–d. The decrease of the frequency and the Vmax-upstroke caused by ryanodine was less in TRPC7 knockdown group. Representative CaTs recorded from the control group g before and h after applying thapsigargin (TG). Representative CaTs recorded from TRPC7 knockdown group i before and j after applying TG. k, l Bar charts showing the normalized frequency and Vmax-decay calculated from g–j. The decrease of the frequency and the Vmax-decay and amplitude caused by TG was less in TRPC7 knockdown group. Representative CaTs recorded from the control group m before and n after applying 2-aminoethoxydipheylborate (2-APB). Representative CaTs recorded from TRPC7 knockdown group o before and p after applying 2-APB. q, r Bar charts showing the normalized frequency and the Vmax-upstroke calculated from m–p. The decrease of the frequency and the Vmax-upstroke caused by 2-APB was similar between control and TRPC7 knockdown groups. Data were presented as mean ± SEM (n = 10 cells; cells were from 3 independent batches of differentiation). *P < 0.05, **P < 0.01 vs control

Fig 3: GFP expression in the brains of adult Tg(eno2:GFP) zebrafish. (A) Parasagittal sections of Tg(eno2:GFP)Pt404 (upper panel) and wild-type (lower panel) brain were labelled with an antibody to GFP. Bound antibody was detected using a histochemical reaction with a red product and nuclei were labelled with a blue counterstain. GFP expression was apparent throughout the brain of transgenic adults. (B) High-power view of medullary reticular formation of Tg(eno2:GFP)Pt404 zebrafish brain, showing GFP expression in cells with neuronal morphology. The arrows show GFP-expressing neurons with axons in the plane of the section. (C–F) Double label confocal images of Tg(eno2:GFP)Pt404 adult brains. Each panel consists of a set of three images: (i) Upper image; red: a cell type-specific marker of interest was localized using specific antibodies. C: Medulla; ChAT, choline acetyltransferase; cholinergic neurons; D: Olfactory bulb; TH, tyrosine hydroxylase; dopaminergic neurons; E: cerebellum; IP3R1, IP3 receptor 1; cerebellar Purkinje cells; D: thalamus; GABA, ?-amino butyric acid; inhibitory neurons; (ii) Middle image, green: GFP expression was localized in the same sections using a GFP antibody; (iii) Lower image: the merged images show co-localization of GFP and the cell type-specific marker (yellow), and were counterstained with DAPI to show nuclei (blue). The scale bar (10 µm) for each set of images is shown in the lower panel.

Fig 4: Components of the spectrin subnetwork co-localize with Kcc2 in several neuronal cellular compartments. Primary cultured neurons from P0 pups were infected with CamKII-AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for Kcc2 and components of the spectrin subnetwork; Sptan1, Sptbn1, Itpr1, and Scn2a. Co-localization was measured using line scans (white lines) and represent the normalized fluorescent intensity of single pixels (approximately 1 µm) on dendrites (n = 3). Scale bars are 10 µm for large images and 5 µm for cropped images.