Journal: Scientific reports

Article Title: Biophysical properties of Na V 1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells.

doi: 10.1038/s41598-023-47310-6

Figure Lengend Snippet: Figure 1. Characterization of specific-markers of vCMs and aCMs. (A) qPCR analysis of several cardiomyocyte genes implicated in cellular excitability (SCN5A, CACNA1C, CACNA1D, GJA1) and contraction (TNNT2, MYL2, MYL7). The analysis of exon 25 of SCN5A mRNA covered all isoforms, including the adult and neonatal isoforms. (B) Fluorescence images showing immunolabeling of cardiac TNNT2, MYL7, MYL2, ACTN1 (α-actinin), GJA1 (connexin 43), and nuclei (DAPI, cyan) (scale bar: 40 µm). Immunofluorescence images were acquired using Zeiss LSM780 confocal microscope, processed with ZEN software (Zeiss), and adapted with ImageJ software version 1.54f (NIH, Bethesda, MD, USA). (C) Western blot analysis of the expression of several excitation–contraction coupling proteins and ion channels in vCMs and aCMs. All images of cropped blot section were exposed with an optimal time to observe protein bands. All cropped blot sections were delimited by black lines. Cropped strain-free blots showing total proteins served as loading control. Original blots are presented in Suppl. Fig. S4. Top panel, middle panel, and bottom panel, respectively, refer to “Blot 1”, Blot 2” and “Blot 3″ in Suppl. Fig. S4. Cropped section areas are indicated in Suppl. Fig. S4 by red lines. Immunoblot images were adapted with ImageJ software and arranged with Microsoft Powerpoint software version microsoft 365 (Microsoft, Redmond, WA, USA).

Article Snippet: The PVDF membranes were blocked and were incubated with rabbit anti-sodium voltage-gated channel alpha subunit 5 (SCN5A) (1:200, Cat# ASC-005, Alomone Labs, RRID:AB_2040001), rabbit anti-calcium voltagegated channel subunit alpha 1 C (CACNA1C) (1:200, Cat# ACC-003, Alomone Labs, RRID:AB_2039771), mouse anti-myosin light chain 7 (MYL7) (1:400, Cat# ab68086, Abcam, RRID:AB_1140497), rabbit anti-myosin light chain 2 (MYL2) (1:2000, Cat# ab79935, Abcam, RRID:AB_1952220), mouse anti-TNNT2 (1:5000, Cat# ab10214, Abcam, RRID:AB_2206574), rabbit anti-gap junction protein alpha 1 (GJA1) (1:5000, Cat# ab11370, Abcam, RRID: AB_297976), rabbit anti-potassium voltage-gated channel subfamily A member 5 (KCNA5) (1:200, Cat# APC-150, Alomone Labs, RRID: AB_10918640), rabbit anti-ryanodine receptor 2 (RYR2) (1:1000, Cat# ARR002, Alomone Labs, RRID: AB_2040184), or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:20,000, Cat# A300-641A, Bethyl, RRID:AB_513619).

Techniques: Fluorescence, Immunolabeling, Immunofluorescence, Microscopy, Software, Western Blot, Expressing, Control

Journal: Journal of cellular physiology

Article Title: Primary Cilium Regulates CaV1.2 Expression Through Wnt Signaling

doi: 10.1002/jcp.24642

Figure Lengend Snippet: shRNA sequences

Article Snippet: Prior to experiments, cells were treated with 100 ng/ml recombinant Wnt3a (R&D Systems, Minneapolis, MN) for 3 days and serum starved for 24 h. RNAi knockdown cells shRNA lentiviral vectors specific to Cacna1c ( Origene ; pGFP-C-shLenti clone ID: TL500242) were transfected into HEK293T cells.

Techniques: shRNA, Control

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: STIM1 deficiency is linked to Alzheimer’s disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca 2+ entry

doi: 10.1007/s00109-018-1677-y

Figure Lengend Snippet: Increased cellular Ca 2+ influx underlies mitochondrial failure and augmented senescence. a Changes in cytosolic-free Ca 2+ concentration were analyzed in fura-2-loaded cells. Cells in HBSS containing 1.26 mM Ca 2+ were subjected to 1-min depolarization with 90 mM KCl (red line). CaCl 2 in the HBSS was increased to 5 mM during depolarization to facilitate the Ca 2+ influx recording. In parallel experiments, 10 μM nifedipine was added to the assay medium during the recording (black line). Right panel: the increase of the F340/F380 ratio triggered by 90 mM KCl in the presence of VOCCs blockers is shown as mean ± s.d. of 3 experiments (a minimum of 70 cells per experimental condition). Final concentrations: 10 μM nifedipine, 1 μM ω-conotoxin MVIIC, 3 μM ML 218. b STIM1-KO cells, or STIM1-KO cells stably expressing a specific shRNA to knock-down CACNA1C transcripts, were treated as described in panel ( a ). The left panel shows a representative experiment, and the bar chart of the right panel shows the increase in the F340/F380 ratio evoked by depolarization (mean ± s.d. of two independent experiments; n > 60 cells per condition). c Senescence (left panel) and mitochondrial polarization (middle and right panels) were assessed from differentiated cells after 6 DIV, staining with C12FDG as described in Fig. c and TMRM as in Fig. b–d, respectively. Data are mean ± s.d. of three independent experiments (number of replicates is shown for each condition). d Rotenone-sensitive NADH oxidase activity was assessed from differentiated SH-SY5Y cell lysates (wild-type, STIM1-KO, and STIM1-KO + shRNA for CACNA1C ). Data are presented as the mean ± s.d. of two independent experiments. e Cell were transiently transfected for the expression of the Ca 2+ sensor 4mtD3cpv. Mitochondrial [Ca 2+ ] was assessed as described in Fig. . Data of six independent experiments are shown in the right panel bar chart as mean ± s.d.

Article Snippet: The construct with the 29mer shRNA cloned into the pRFP-C-RS plasmid to knock-down CACNA1C gene expression was purchased from OriGene (#TF314247-A).

Techniques: Concentration Assay, Stable Transfection, Expressing, shRNA, Knockdown, Staining, Activity Assay, Transfection