Journal: Frontiers in Cardiovascular Medicine

Article Title: Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression

doi: 10.3389/fcvm.2022.763544

Figure Lengend Snippet: CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.

Article Snippet: Transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against mouse monoclonal CCN5 (1:1,000, Sigma, #WH0008839M9), mouse monoclonal α-SMA (1:1,000, Sigma-Aldrich, #A5228), goat polyclonal collagen I (1:1,000, Abcam, ab34710), rabbit polyclonal SERCA2a (1:1,000, a custom antibody from 21st Century Biochemicals), rabbit polyclonal RyR2 (1:1,000, Alomone Lab, ARR-002), rabbit monoclonal NCX1 (1:1,000, Abcam, EPR12739), rabbit polyclonal p-PLN (1:1,000, Badrilla, A010-12AP), mouse monoclonal t-PLN (1:1,000, Badrilla, A010-14), and mouse monoclonal α-tubulin (1:3,000, Santa Cruz, #sc-8035) for 12–16 h at 4°C.

Techniques: Injection, Staining, Western Blot

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

Article Snippet: Rabbit polyclonal anti RyR2 , Alomone , Cat# ARR-002; RRID: AB_2040184.

Techniques: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti RyR2 , Alomone , Cat# ARR-002; RRID: AB_2040184.

Techniques: Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats

doi: 10.3390/ijms21249593

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti-Ryanodine Receptor 1 , Ryanodine Receptor 1/ICC , Rabbit polyclonal , 1:200 , Alomone Labs.

Techniques: Marker

Journal: Nature Communications

Article Title: Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm

doi: 10.1038/s41467-017-00127-0

Figure Lengend Snippet: Structural changes consequent to PKP2 deletion. a STORM-acquired images of Cav1.2 ( green ) and RyR2 ( purple ) in a single myocyte. b Analysis of RyR2/Cav1.2 overlapping area in transverse ( left ) and longitudinal ( right ) clusters. n = 1335 and 1285 clusters from 30 control and 25 PKP2-cKO cardiomyocytes at 21 dpi, respectively. t -test, * p < 0.05 and ** p < 0.01 vs. control. c 2D-EM image of PKP2-cKO ventricular tissue at 21 dpi showing a dyadic structure. Scale bar = 200 nm. d Boundaries of the jSR ( red ) and T-tubule ( blue ) membranes, detected from the dyad in the dotted square in c . e Acquisition of distances from each point in the T-tuble membrane to its closest neighbor in the jSR ( green lines ). f Close-up of the region inside the dashed square in e . All distances measured within a dyad were averaged to obtain the “average distance” for that dyad (expressed in nm). g Comparison of average data collected from one control ( n = 30 dyads) and 2 PKP2-cKO 21 dpi samples ( n = 41 dyads for both). Student’s t -test * p < 0.001 vs. control. h Spatial orientation of the T-tubular network, obtained from segmentation and analysis of a volume of 15 × 12 × 0.8 µm 3 dimensions obtained by Serial Block-Face Scanning Electron Microscopy analysis. See also Supplementary Video and “Methods”. The angle of the T-tubular skeleton is color-coded ( bottom left ) from −90° in magenta to +90° in red ; relative to the longitudinal axis of the cell ( light blue ; 0°). i Histogram of orientations (from h ), showing a strong preference for zero-degree orientation, as expected from a non-failing heart (see ref. )

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-PKP2 (K44262M; Biodesign International, Meridian Life Sciences), monoclonal mouse anti-AnkyrinB (N105-17; BioLegend), polyclonal rabbit anti-Cav1.2 (ACC-003; Alomone), polyclonal rabbit anti-CASQ2 (PA1-913; ThermoFisher), monoclonal mouse anti-RyR2 (MA3-916; ThermoFisher), polyclonal rabbit anti-RyR2 phospho serine-2808 and anti-RyR2 phospho serine-2814 (A010-30 and A010-31 respectively; Badrilla), monoclonal mouse anti-TriadinT32 (MA3-927; ThermoFisher), polyclonal rabbit anti-NCX (sc-32881, SantaCruz), polyclonal rabbit anti-SERCA2 (PA5-29380; ThermoFisher), monoclonal mouse anti-phospholamban (sc-393990; SantaCruz), polyclonal rabbit anti-phospholamban phospho serine-16 (sc-12963; SantaCruz) and phospho threonine-17 (sc-17024; SantaCruz), GAPDH (G109A; Fitzerald) monoclonal mouse anti-Cx43 (Clone 4E6.2, Millipore), polyclonal rabbit anti-β-catenin (C2206; Sigma), monoclonal mouse anti-JPH2 (sc-37086, SantaCruz), monoclonal mouse anti-Bin1 (Clone 99D; Sigma), polyclonal rabbit anti-PKC and anti-phospho PKC (#2056 and #9375; Cell Signaling), polyclonal rabbit anti-CaMKII (PA5-22168; ThermoFisher), monoclonal mouse anti-CaMKII phospho threonine-286 (MA1-047; ThermoFisher), polyclonal rabbit Desmocollin-2 (ab72792; Abcam), polyclonal rabbit anti-Nav1.5 (S0819; Sigma).

Techniques: Blocking Assay, Electron Microscopy