Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 1. Protein expression of RyR1 and DHPR in muscle biopsy of patients with different RYR1 mutations. Double staining of RyR1 and DHPR, and NADH staining in muscle biopsies of patients with congenital myopathy and RYR1 mutations. NADH staining was performed in non-serial sections from RyR1/DHPR double staining. Scale bar = 25 µm.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Expressing, Double Staining, Staining

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 2. Quantitative reverse transcriptional real-time PCR of RYR1 and CACNA1S in skeletal muscle biopsies from patients carrying different RYR1 mutations. The relative content of RYR1 and CACNA1S mRNA was assessed by real-time PCR using Ct method and using DES as muscle specific reference gene. The relative expression of RYR1 and CACNA1S to DES in one of the control individuals was set as 1.0, and was used to calibrate the values of all other samples. Each symbol represents an individual and the bar indicates mean relative expression. ANOVA was used for the statistical analysis of the values in different groups, followed by the Bonferroni post statistics test. A: Relative expression of RYR1 transcript in muscle biopsies. B: Relative expression of CACNA1S transcript in muscle biopsies. ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 3. RYR1, CACNA1S, and DES mRNA expression in myotubes treated with RYR1 siRNA. The relative quantification of RYR1 (A), CACNA1S (B), and DES (C) mRNA was performed by quantitative real-time PCR. The value was obtained from three independent transfection experiments of myotubes treated with RYR1 siRNA at concentrations of 10, 30, and 50 nM. The data are presented as mean ± SEM, N = 3. ANOVA was used for the statistical analysis of the values in the RYR1 siRNA-treated myotubes to negative control siRNA, followed by Bonferroni post statistics test. The relative expression of target genes in control siRNA-treated myotubes was set as 1.0, and was used to normalize the values in the RYR1 siRNA-treated myotubes. ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 4. Downregulation of RyR1 by RYR1 siRNA transfection does not affect myotube differentiation: Cells were transfected with 50 nM RYR1 siRNA, differentiated for 5 days and then stained with β-tubulin and DAPI, the diameter of myotubes with >3 nuclei were measured and averagedconsidered. A: Mean (±SE) myotube diameter (µm) of 30 transfected and 30 mock transfected myotubes. (B: Relative mRNA expression of differentiation-related markers; Dystroglycan 1 (DAG), Myosin heavy chain 1 (MYH1), Troponin T1 (TNNT1), and Desmin (DES) normalized to GAPDH expression as internal control (N = 6). C: Immunofluorescentce of transfected (RYR1siRNA) or mock transfected (control) myotubes stained with anti- β tubulin (green) and DAPI and observed with a Nikon A1R confocal microscope with a 40× NeoFluar objective (1.4 NA). Bar indicates 30 µm.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Transfection, Staining, Expressing, Control, Microscopy

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 5. RyR1, DHPR, and other SR protein in RYR1 siRNA-treated myotubes and muscle biopsies from patients with RyR1 deficiency due to recessive RYR1 mutations. A: Representative western blots of RyR1, DHPR, IP3R-III, α-actinin 2, SERCA 2, desmin, and β-tubulin in human myotubes treated by RYR1 siRNA. B: Semi-quantification of proteins in RYR1 siRNA-treated myotubes. The expression of proteins was normalized to tubulin. Protein content in RYR1 siRNA-treated group was compared with the control group. C: Representative western blots of RyR1, DHPR, IP3R-III, α-actinin 2, SERCA 2, and desmin in muscle biopsies from two patients (patient 2 and 5 in Supp. Table S1) with recessive RYR1 mutations. D: Semi-quantification of proteins in two patients, with RyR1 deficiency due to recessive RYR1 mutations, and three controls. The expression of proteins was normalized by desmin.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Western Blot, Expressing, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 6. The expression of ITPR1, ITPR2, and ITPR3 mRNA in RYR1 siRNA-treated myotubes and skeletal muscle biopsies from patients with different RYR1 mutations. A: The relative expression of ITPRs mRNA in cultured myotubes treated with siRNA was measured by quantitative reverse transcript real-time PCR. Data are presented as mean ± SEM. N = 4 samples per group. ∗P < 0.05. B: The relative expression of ITPRs mRNA measured by quantitative real-time PCR in skeletal muscle biopsies from controls (N = 7), patients with dominant RYR1 mutations (N = 5) and patients with complex recessive RYR1 mutations with RyR1 protein deficiency (N = 6). GAPDH was used as general internal control gene. C: The relative expression of ITPRs mRNA measured by quantitative real-time PCR in skeletal muscle biopsies by using DES as muscle-specific internal control gene.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 7. Analysis of calcium regulation in a human muscle cell line after transfection with RYR1 siRNA. Cells were transfected with 50 nM RYR1 siRNA or mock transfected as described in the Materials and Methods section and were either untreated or treated with 1 µM Xestospongin C during the 40 min of fura-2 loading. Myotubes were stimulated with the indicated agonist in Krebs-Ringer containing no added Ca2+ plus 100 µM La3+ and the total amount of calcium released was calculated using Origin software by calculating the total transient, i.e. the area under the curve. Resting [Ca2+] is presented as fluorescence ratio (340/380 nm) (au) before cell stimulation in Krebs-Ringer containing 2 mM Ca2+; the total amount of rapidly releasable Ca2+ present in the SR/ER stores was obtained by calculating the area under the curve after exposing cells to 1 µM ionomycin in the Krebs-Ringer containing no added Ca2+ plus 0.5 mM EGTA. For details see Materials and Methods section. All results are expressed as mean value (±SEM) of the indicated number of cells. White bars mock transfected cells, grey bars cells transfected with RYR1 siRNA. Statistical analysis was performed using Student’s t-test.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Transfection, Software, Cell Stimulation

Journal: bioRxiv

Article Title: Multiscale activity imaging in mammary gland reveals how oxytocin enables lactation

doi: 10.1101/657510

Figure Lengend Snippet: ( A ) Ryr1 , Ryr2 and Ryr3 expression in lysates prepared from whole mammary tissue (including luminal, basal and stromal cells) dissected from virgin or lactating animals (n = 4 mice). ( B ) Krt14 , Esr1 and Ryr1 levels in freshly sorted luminal and basal cells (n = 3 mice). Graphs show mean ± SEM; * P < 0.05 (Student’s t-test); n.d., not detected. Ryr2 and Ryr3 transcripts were either not detected or detected at very low levels in only a fraction of samples from both luminal and basal cells.

Article Snippet: The following Gene Expression Assays were used in this study: Ryr1 (Mm01175211_m1), Ryr2 (Mm00465877_m1), Ryr3 (Mm01328395_m1), Krt14 (Mm00516876_m1) and Esr1 (Mm00433149_m1).

Techniques: Expressing