Journal: bioRxiv

Article Title: Dual effects of nicotinamide on aging-related arrhythmia: protective at low dose, proarrhythmic at high doses

doi: 10.1101/2025.09.22.677785

Figure Lengend Snippet: (A) Experimental scheme of Langendorff ex vivo mouse hearts perfused with 0 mM NAM (control) or 100 mM NAM-supplemented buffer. (B) Representative electrocardiogram (ECG) shows that acute perfusion with 100 mM NAM rapidly induced VF, contractile dysfunction, and eventually led to cardiac arrest (n=4 males, 2 females). (C) Representiative blots and (D) quantification of acetylated SERCA2a levels of left venticular tissue from control and 100 mM NAM-perfused ex vivo mouse hearts (n=3). Data are expressed as mean ± standard error of the mean. Two tailed unpaired Student t-test was used.

Article Snippet: The membrane was then blocked with Intercept Blocking Buffer (927-60001, LI-COR) for 1 h at RT, and incubated with primary antibodies against SERCA2a (1:1000 dilution, 4388, cell signaling), and Pan Acetylation (1:2000 dilution, 66289-1-Ig, Proteintech) overnight at 4°C.

Techniques: Ex Vivo, Control, Two Tailed Test

Journal: Diabetologia

Article Title: SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells.

doi: 10.1007/s00125-023-05979-4

Figure Lengend Snippet: Fig. 3 Beta cell-specific SERCA2 deficiency results in an increase in serum and total pancreas P/I ratios and alters the expression of prohormone convertase isoforms in islets. (a–c) Blood was collected from randomly fed control and βS2KO mice at 25 weeks of age and serum was isolated. Serum levels of insu- lin (a) and proinsulin (b) were determined by ELISA and the P/I ratio (c) was calculated (n=12–13 mice). (d–f) Total protein was extracted from the whole pancreas and the levels of insulin (d) and proinsulin (e) were determined by ELISA and the P/I ratio

Article Snippet: Antibodies against SERCA2 (goat, cat. no. sc-8095; RRID:AB_2290108), SERCA2 in ESM Figs 4 and 5 (mouse, cat. no. sc-376235; RRID:AB_10989947) and actin in ESM Figs 4 and 5 (mouse, cat. no. sc-47778; RRID:AB_626632) were from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Diabetologia

Article Title: SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells.

doi: 10.1007/s00125-023-05979-4

Figure Lengend Snippet: Fig. 4 Enzyme activity of prohormone convertases is regulated in a SERCA2-dependent manner in beta cell lines. (a) Representative immunoblots of SERCA2, PC1/3, PC2, CPE, proinsulin and insulin in WT and S2KO cells transduced with empty adenovirus or an ade- novirus expressing human SERCA2b (Ad-SERCA2). (b–e) Quanti- fication of immunoblotting results: (b) PC1/3, (c) proPC2, (d) PC2 and (e) CPE. Expression of proteins was normalised to actin expres- sion (n=6–9). (f) Quantification of the P/I ratio (n=6). (g, h) WT and S2KO INS-1 cells were transduced with empty adenovirus or Ad-SERCA2 and the enzymatic activity of secreted PC1/3 and PC2 was determined using fluorometric assays. Enzyme activity was nor-

Article Snippet: Antibodies against SERCA2 (goat, cat. no. sc-8095; RRID:AB_2290108), SERCA2 in ESM Figs 4 and 5 (mouse, cat. no. sc-376235; RRID:AB_10989947) and actin in ESM Figs 4 and 5 (mouse, cat. no. sc-47778; RRID:AB_626632) were from Santa Cruz Biotechnology.

Techniques: Activity Assay, Western Blot, Transduction, Expressing

Journal: Diabetologia

Article Title: SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells.

doi: 10.1007/s00125-023-05979-4

Figure Lengend Snippet: Fig. 5 Bulk RNA-seq and chemical inhibition of vesicle traf- ficking demonstrate a role for SERCA2 in prohormone matura- tion. (a, b) RNA isolated from islets from 17-week-old control and βS2KO mice was subjected to bulk RNA-seq. (a) Volcano plot indicating differentially expressed genes (FC ≥1.5, p<0.05). Red indicates upregulated genes, blue indicates downregulated genes and black indicates no change in expression in βS2KO islets vs control islets. (b) Dot plot representing the top 20 most relevant GO terms identified using the ggplot2 package in R. The size of the dots indicates the number of genes in each term.

Article Snippet: Antibodies against SERCA2 (goat, cat. no. sc-8095; RRID:AB_2290108), SERCA2 in ESM Figs 4 and 5 (mouse, cat. no. sc-376235; RRID:AB_10989947) and actin in ESM Figs 4 and 5 (mouse, cat. no. sc-47778; RRID:AB_626632) were from Santa Cruz Biotechnology.

Techniques: RNA Sequencing, Inhibition, Isolation, Control, Expressing

Journal: Diabetologia

Article Title: SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells.

doi: 10.1007/s00125-023-05979-4

Figure Lengend Snippet: Fig. 6 SERCA2 loss disrupts protein trafficking within the beta cell secretory pathway. (a–c) WT and S2KO INS-1 cells were treated with 10 mol/l cycloheximide with or without 10 µmol/l MG132 for 9 h prior to protein extraction. (a) Representative immunoblots of the 87 kD less active form of PC1/3 and proPC2. (b, c) Quanti- fication of time-dependent changes in the levels of 87 kD PC1/3 (b) and 70–75 kD proPC2 (c) after the addition of MG132 (n=3) independent experiments. (d, e) Pancreatic sections from control and βS2KO mice were stained for proinsulin, insulin and DAPI. (d) Representative immunofluorescent images and (e) quantification of protein expression levels using ImageJ (n=5). (f–h) Pancreatic

Article Snippet: Antibodies against SERCA2 (goat, cat. no. sc-8095; RRID:AB_2290108), SERCA2 in ESM Figs 4 and 5 (mouse, cat. no. sc-376235; RRID:AB_10989947) and actin in ESM Figs 4 and 5 (mouse, cat. no. sc-47778; RRID:AB_626632) were from Santa Cruz Biotechnology.

Techniques: Protein Extraction, Western Blot, Control, Staining, Expressing

Journal: Diabetologia

Article Title: SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells.

doi: 10.1007/s00125-023-05979-4

Figure Lengend Snippet: Fig. 7 Decreased expression of active prohormone convertase iso- forms in GLT-treated mouse and human islets. (a–j) Isolated WT mouse islets were subjected to GLT treatment (25 mmol/l glucose + 0.5 mmol/l palmitate) for 24 h. (a) Representative immunoblots of SERCA2, PC1/3, PC2, CPE, proinsulin and insulin and (b–j) quan- tification of immunoblotting results: (b) SERCA2, (c) insulin, (d) P/I ratio, (e) proinsulin, (f) PC1/3 87 kDa, (g) PC1/3 66 kDa, (h) proPC2, (i) PC2, (j) CPE. Expression of proteins was normalised to actin expression and FC is relative to the mean control value (n=4–

Article Snippet: Antibodies against SERCA2 (goat, cat. no. sc-8095; RRID:AB_2290108), SERCA2 in ESM Figs 4 and 5 (mouse, cat. no. sc-376235; RRID:AB_10989947) and actin in ESM Figs 4 and 5 (mouse, cat. no. sc-47778; RRID:AB_626632) were from Santa Cruz Biotechnology.

Techniques: Expressing, Isolation, Western Blot, Control

Journal: JCI Insight

Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

doi: 10.1172/jci.insight.170739

Figure Lengend Snippet: ( A ) Immunoblot of SERCA2 in lysates from ATP2A2 wild-type (WT, +/+), heterozygous (HET, +/-), and homozygous knockout (KO, -/-) cells. hTERT-immortalized human epidermal keratinocytes (THEKs) were differentiated in E-medium for 72 hours before lysate harvesting; data represent 3 independent experiments; and β-actin is a loading control. ( B ) Immunofluorescence of SERCA2 (green) in WT, HET, and KO THEKs; images are representative of 14 independent high-powered fields (hpf) per genotype; Hoechst (blue) stains nuclei; scale bar = 10 μm. ( C ) Mechanical dissociation of monolayers from control (+/+), HET (+/-), and KO (-/-) cells grown in 1.3 mM CaCl 2 for 72 hours prior to using dispase to release intact monolayers; representative images of fragmented monolayers transferred into 6-well cell culture plates after mechanical stress are shown. ( D ) Graphs display mean ± SD of the number of epithelial fragments with data plotted for N = 3 biological replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons. ( E ) Change (Δ) in intensity of GCaMP in control (+/+), HET (+/-), and KO (-/-) THEKs from baseline at t = 0 seconds with addition of 1 mM CaCl 2 at t = 60 seconds (arrow); data plotted as mean ± SEM from N = 3 independent experiments per genotype. ( F ) Representative fluorescence images of GCaMP in WT, HET, and KO cells in low CaCl 2 (left) or high CaCl 2 (right); scale bar = 10 μm.

Article Snippet: Rabbit antibodies against SERCA2 (D51B11; catalog 9580) and p-ERK1/2 (D13.14.4E; catalog 4370) as well as mouse antibodies against ERK1/2 (L34F12; catalog 4696) and β-actin (8H10D10; catalog 3700) are from Cell Signaling Technology.

Techniques: Western Blot, Knock-Out, Immunofluorescence, Cell Culture, Fluorescence

Journal: JCI Insight

Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

doi: 10.1172/jci.insight.170739

Figure Lengend Snippet: ( A ) Results from bulk RNA sequencing of SERCA2 HET versus control cells. Volcano plot depicts log 2 fold-changes of genes significantly downregulated (blue) and upregulated (red) with cutoff (dashed line) 0.05 for adjusted P value. ( B ) Gene ontology (GO) analysis of transcripts significantly altered (adjusted P ≤ 0.05) in HET cells revealed upregulation (red) in genes controlling ER stress and growth factor signaling and downregulation (blue) of genes controlling epidermal differentiation and antiviral response. ( C ) Control (+/+), HET (+/-), or KO (-/-) THEKs were differentiated in E-medium for 24 hours, and mRNA transcripts were quantified by reverse transcription quantitative PCR (RT-qPCR). Graphs display mean ± SD with plotted values from N = 4 biological replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons. DSG1, desmoglein 1; KRT, keratin.

Article Snippet: Rabbit antibodies against SERCA2 (D51B11; catalog 9580) and p-ERK1/2 (D13.14.4E; catalog 4370) as well as mouse antibodies against ERK1/2 (L34F12; catalog 4696) and β-actin (8H10D10; catalog 3700) are from Cell Signaling Technology.

Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: JCI Insight

Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

doi: 10.1172/jci.insight.170739

Figure Lengend Snippet: ( A ) Control or HET cells were differentiated in E-medium ± MEK inhibitors for 24 hours, and then transcripts were measured by RT-qPCR; graphs show mean ± SD from N = 4 replicates; P values from ANOVA (Brown-Forsythe) with Dunnett’s T3 adjustment for multiple comparisons. ( B ) Immunostaining KRT10 (and Hoechst) in control or HET cells treated with DMSO or 1 μM trametinib (Tram) for 48 hours; images represent 2 independent experiments using 2 control and 2 SERCA2-deficient lines; scale bar = 50 μm. ( C ) Quantification of KRT10 immunostaining in control versus SERCA2-deficient cells treated with DMSO or 1 μM Tram for 24 hours; data shown as a box plot of 25th–75th percentile with line at the median from N ≥ 21 nonoverlapping hpf per group; control mean normalized to 1; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( D ) Immunoblot of KRT10 and KRT14 (β-actin loading control) in lysates from control versus HET cells treated with DMSO, 1 μM dabrafenib (Dab), or MEK inhibitors (1 μM Tram; 10 μM U0126; 10 μM PD184; 20 μM PD980); data represent 2 independent experiments. ( E ) Quantification of fragments of HET and KO monolayers treated with DMSO or 1 μM trametinib for 48 hours; graphs display mean ± SD for N = 3 replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( F ) Images of 6-well culture plates containing fragmented monolayers of control or HET cells treated with DMSO or 20 μM PD980 for 24 hours. ( G ) Quantification of fragments of control, HET, or KO monolayers treated with DMSO or 20 μM PD980 for 24 hours; graphs display mean ± SD for N = 3 replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons.

Article Snippet: Rabbit antibodies against SERCA2 (D51B11; catalog 9580) and p-ERK1/2 (D13.14.4E; catalog 4370) as well as mouse antibodies against ERK1/2 (L34F12; catalog 4696) and β-actin (8H10D10; catalog 3700) are from Cell Signaling Technology.

Techniques: Quantitative RT-PCR, Immunostaining, Western Blot

Journal: JCI Insight

Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

doi: 10.1172/jci.insight.170739

Figure Lengend Snippet: ( A ) Immunostaining of KRT10 (and Hoechst) in THEKs treated with DMSO, 1 μM TG, or 1 μM TG plus 1 μM Tram (TG + Tram) for 48 hours; scale bar = 50 μm. ( B ) Quantification of KRT10 immunostaining in THEKs treated with DMSO, TG, or TG plus Tram; data shown as a box plot of the 25th–75th percentile with a line at the median from N ≥ 31 nonoverlapping hpf from 2 independent THEK lines for both control and SERCA2-deficient cells; control mean normalized to 1; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( C ) Representative images of fragmented monolayers transferred into 6-well cell culture plates are shown for NHEKs treated with DMSO versus 1 μM TG alone or with 1 μM dabrafenib versus an MEK inhibitor (1 μM Tram, 10 μM U0126, or 20 μM PD980) for 24 hours. ( D ) Quantification of epithelial fragments of NHEK monolayers; bar graphs display mean ± SD with data points for N = 6 biological replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( E ) H&E-stained tissue cross sections of organotypic cultures of NHEKs treated with DMSO, 1 μM TG, or 1 μM TG plus 20 μM PD980, the latter displaying improved keratinocyte cohesion and normalization of cornification; scale bar = 100 μm. ( F ) Quantification of retained nuclei in cornified layers of organotypic cultures treated with the indicated inhibitors for 48 hours; graph displays mean ± SD with plotted values averaged from ≥49 nonoverlapping hpf per condition from N = 4 biological replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons.

Article Snippet: Rabbit antibodies against SERCA2 (D51B11; catalog 9580) and p-ERK1/2 (D13.14.4E; catalog 4370) as well as mouse antibodies against ERK1/2 (L34F12; catalog 4696) and β-actin (8H10D10; catalog 3700) are from Cell Signaling Technology.

Techniques: Immunostaining, Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Ryanodine Receptor Staining Identifies Viable Cardiomyocytes in Human and Rabbit Cardiac Tissue Slices

doi: 10.3390/ijms241713514

Figure Lengend Snippet: Methodological approach. ( A ) Human and rabbit myocardial slices were created from left-ventricular transmural samples with a vibratome and then cultivated for up to seven days in biomimetic cultivation chambers containing stimulation electrodes and a force transducer. Subsequently, dextran-based death staining was applied, followed by chemical fixation, fluorescent staining, confocal microscopy and image processing. ( B ) Example image of a rabbit cardiac slice stained with fixable, fluorescent dextran (green), for ryanodine receptors (RyR, red) and with wheat germ agglutinin (WGA, blue). ( C ) Histogram-based local thresholds were applied to identify the dextran-positive (green) and RyR-positive (red) pixels, as well as ( D ) the WGA-positive pixels (white). ( E ) Using the WGA distance map, a watershed transform was applied to segment individual myocytes (different colors). ( F ) For each cell segment, the number of RyR- and dextran-positive pixels was counted. If the fraction of these pixels within the segment exceeded a defined threshold, segments were classified as RyR-positive, i.e., living, myocytes (magenta) and dextran-positive, i.e., dead, myocytes (green). Scale bar length in ( B ) is 20 µm and also applies to ( C – F ).

Article Snippet: For immunostaining, the slices were incubated with primary antibody against cardiac ryanodine receptor (IgG1, mouse, C3-33, Thermo Fisher, Braunschweig, Germany) or SERCA2 (IgG2a, mouse, 2A7-A1, Thermo Fisher) 1:200 in blocking solution (BS: 5% NGS, 5% BSA, 0.25% Triton-X in PBS) for 4 h at RT or overnight at 4 °C.

Techniques: Staining, Confocal Microscopy