Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Transcriptional Studies on Trypanosoma cruzi – Host Cell Interactions: A Complex Puzzle of Variables

doi: 10.3389/fcimb.2021.692134

Figure Lengend Snippet: Different T. cruzi -host cell interaction studies incorporating transcriptomics of the infected host cell.

Article Snippet: 4 , Dm28c/TcI , Vero , Primary human cardiomyocytes (CelProgen) , 10:1 , 2h , 0, 3, 6, 12hpi , Microarrays (Agilent) , Up-regulation of OXPHOS related genes ( ) .

Techniques: Infection, In Vitro, Genome Wide, Sequencing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Transcriptional Studies on Trypanosoma cruzi – Host Cell Interactions: A Complex Puzzle of Variables

doi: 10.3389/fcimb.2021.692134

Figure Lengend Snippet: Transcriptomic and functional results from different studies related to the host cell respiration response to T. cruzi infection.

Article Snippet: 4 , Dm28c/TcI , Vero , Primary human cardiomyocytes (CelProgen) , 10:1 , 2h , 0, 3, 6, 12hpi , Microarrays (Agilent) , Up-regulation of OXPHOS related genes ( ) .

Techniques: Functional Assay, Infection, In Vitro, In Vivo, Sequencing, Staining, Inhibition, Western Blot, Immunohistochemistry

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Transcriptional Studies on Trypanosoma cruzi – Host Cell Interactions: A Complex Puzzle of Variables

doi: 10.3389/fcimb.2021.692134

Figure Lengend Snippet: Studies related to the host AKT/PI3K response to T. cruzi infection.

Article Snippet: 4 , Dm28c/TcI , Vero , Primary human cardiomyocytes (CelProgen) , 10:1 , 2h , 0, 3, 6, 12hpi , Microarrays (Agilent) , Up-regulation of OXPHOS related genes ( ) .

Techniques: Infection, Activity Assay, Western Blot, Expressing, Activation Assay, Genome Wide, Over Expression, In Vitro, In Vivo

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, b, Representative fields of heart cultures stained with DAPI (blue), cTnT (green) and Ki67 (red). White arrows indicate Ki67+cTnT+ cells. CMs, cardiomyocytes. c, d, Percentage of proliferating cardiomyocytes from P1 (c) or P7 (d) hearts in response to P1 and P7 ECM particles. n = 2,069 cardiomyocytes from three samples (c); n = 2,221 cardiomyocytes from four samples (d). e, f, Percentage of proliferating cardiomyocytes (Ki67+cTnT+) in response to P1 and P7 ECM in P1 (e) or P7 (f) cultures, with or without the broad MMP inhibitor (Marimastat). n = 3,480 cardiomyocytes from three samples (e); n = 23,445 cardiomyocytes from four samples (f). g, h, Percentage of P1 (g) or P7 (h) proliferating cardiomyocytes in response to MMP9- or MMP12-cleaved ECM fragments. n = 11,820 cardiomyocytes from four samples (g); n = 15,509 cardiomyocytes from four samples (h). i, qPCR of Agrn mRNA in P1 and P7 hearts. n = 8 P1 and 3 P7 hearts. j, Quantification of western blots for agrin from P1, P7 and 12-week-old (12W) adult heart lysates. A.U., arbitrary units. n = 3 samples per group. k, Images of P1 and P7 heart sections stained for agrin (green) and DAPI (blue). n = 3 samples per group. l, qPCR analysis of cardiac populations (FB, fibroblasts; CM, cardiomyocytes; EC, endothelial cells). n = 4 cardiomyocyte, 4 non-cardiomyocyte, 4 fibroblast, 4 non-fibroblast, 7 endothelial cells and 7 non-endothelial cell samples. Scale bars, 40 μm (a) and 10 μm (k). Data are presented as mean ±s.e.m. *P < 0.05, **P< 0.01, ***P< 0.001; statistical significance was calculated using ANOVA followed by a Dunnett’s post hoc test relative to the control group (c–h) or a Tukey’s post hoc test (j), statistical significance was calculated using a one-tailed t-test (i, l).

Article Snippet: Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Control, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, Diagram showing the mesoderm conditional knockout of agrin (agrin-cKO) in mice. b, qPCR of Agrn mRNA in P1 wild-type and agrin-cKO hearts. n = 8 wild-type (WT) and 7 cKO samples. c, Immunofluorescence images of agrin in P1 wild-type and agrin-cKO heart sections. n = 3 samples of each group. d, Quantification of western blots for agrin from wild-type and agrin-cKO mice heart lysates. n = 8 samples of each group. e, Immunofluorescence analysis and Pearson’s correlation coefficient analysis of T-tubules labelled with Cav3 in the z-lines (as indicated by Actn2). n = 4 wild-type and 3 cKO samples. White arrow heads indicate colocalization of T-tubules and z-lines. f, Myh6:Myh7 protein ratio from wild-type and agrin-cKO mice. n = 5 wild-type and 6 agrin-cKO samples. g, Staining and mean pixel intensity quantification of mitochondrial content in cardiomyocytes measured by Tom20 staining. n = 5 wild-type and 3 agrin-cKO samples. h, i, In vivo evaluation of P1 cardiomyocyte cell-cycle markers (Ki67; h) and (Aurkb; i) by immunofluorescence analysis in wild-type and agrin-cKO left ventricle heart sections. n = 41,695 cardiomyocytes from 11 wild-type and 4 agrin-cKO samples (h); n = 3,212 cardiomyocytes from 3 samples per group (i). j, Histological sections of P1 resected wild-type and agrin-cKO mice, 28 days after injury, stained with Masson’s trichrome. Bottom left corner of the cKO (left panel) was cropped to remove the adjacent section. k, l, Scar quantification of heart sections four weeks after resection of wild-type and agrin-cKO hearts. LV, left ventricle; none, 0% of the left ventricular wall; moderated ≤1% of the left ventricular wall; large ≥1% of the left ventricular wall. n = 12 wild-type and 8 cKO mice. m, n, Functional cardiac recovery measurements (ejection fraction (EF) and fractional shortening (FS)) of hearts from agrin-cKO and wild-type mice 28 days after resection. n = 5 wild-type and 3 cKO mice. o, p, In vivo evaluation of cardiomyocyte cell-cycle re-entry in the peri-infarct region by immunofluorescence analysis of Ki67 (o) or Aurkb (p) in heart sections 7 days after resection of wild-type and agrin-cKO hearts. n = 5,556 cardiomyocytes from seven wild-type and six cKO samples (o); n = 2,235 cardiomyocytes from three wild-type and four cKO samples (p). Scale bars, 10 μm (c, e, g–i, p), 20 μm (o), 100 μm (j, right) and 1 mm (j, left). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using a one-tailed t-test.

Article Snippet: Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Knock-Out, Immunofluorescence, Western Blot, Staining, In Vivo, Functional Assay, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a–c, In vivo evaluation of cardiomyocyte cell-cycle re-entry in the peri-infarct region seven days after myocardial infarction by Ki67 (a), Aurkb (b) or 21 days after myocardial infarction BrdU (c) n = 1,842 cardiomyocytes from five PBS and six agrin samples; n = 2,259 cardiomyocytes from five samples per group (b); n = 9,307 cardiomyocytes from six samples per group (c). For BrdU pulse-chase experiment see Methods. d, e, Heart section scar assessment following PBS or agrin treatment at indicated days after myocardial infarction (MI). Representative images are shown in d and quantified in e. n = 4 mice per group for day 0, 5 PBS- and 4 agrin-treated mice for day 4, 4 PBS- and 5 agrin-treated mice for day 14, 7 PBS- and 8 agrin-treated mice for day 35. f–h, Serial echocardiographic measurements of ejection fraction, fractional shortening and wall thickness of uninjured and injured hearts treated with PBS or agrin. LVAW, left ventricle anterior wall; LVPW, left ventricle posterior wall. n = 8 baseline, 5 uninjured, 6 PBS- and 8 agrin-treated mice (f, g); n = 2 uninjured, 5 PBS- and 6 agrin-treated mice (h). Scale bars, 10 μm (a, c), 20 μm (b) and 1 mm (d). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01; statistical significance was calculated using ANOVA followed by Dunnett’s post hoc test relative to control group (f–h) or using a one-tailed t-test relative to PBS (a–e).

Article Snippet: Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: In Vivo, Pulse Chase, Control, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, Western blot of Dag1 and MuSK from P1 and P7 heart lysates. n = 3 samples per group for MuSK, n = 6 samples per group for Dag1. Skeletal muscle extract was used as positive control (PC) for MuSK expression (lower panel). b, c, P7 Dag1 expression by qPCR (b) or membrane staining (c) in cardiomyocytes relative to non-cardiomyocytes (z–x plane presented). WGA, wheat-germ agglutinin. n = 2 cardiomyocyte and 2 non-cardiomyocyte RNA samples; n = 17,012 cardiomyocytes from four samples per group (c). d, e, Western blots of phosphorylated ERK (pERK) and total ERK (tERK) in P7 cultures with the indicated treatments. Ab, antibody. n = 5 samples (d); n = 3 samples (e). f, g, P7 cardiomyocytes cell-cycle analysis by immunofluorescence staining following agrin treatment with a MEK inhibitor (f) or Dag1 inhibitory antibody (g). Ctrl, control; PD, PD0325901. n = 2,743 cardiomyocytes from six samples (f); n = 6,649 cardiomyocytes from four samples (g). h, Glycerol-gradient fractionation of whole-cell extracts from P7 cells with or without (time 0) agrin treatment for 2 and 48 h. Fractions were analysed by SDS–PAGE and immunoblotting (IB) with indicated antibodies. n = 3 samples. Rec-agrin, recombinant agrin. Arrowheads indicate glycosylated form of β-dystroglycan. i, Isolated myofibrillar pellets and cytosol from P7 cells treated with agrin for 48 h analysed by SDS–PAGE and immunoblotting. n = 3 samples. j, Quantification of cardiomyocyte dedifferentiation using Myh6-lineage-derived cardiomyocytes (red mostly nuclear staining) stained with cTnT (green). Arrows indicate cardiomyocytes that have lost cTnT expression. n = 2,869 cardiomyocytes from five samples per group. k, Co-immunoprecipitation (IP) assay of Yap, agrin and various DGC proteins immunoprecipitated with syntrophin. n = 3 samples.l, Co-immunoprecipitation assay from 0, 2, 24 and 48 h agrin-treated cell membranes immunoprecipitated by syntrophin and blotted for Yap, agrin and other DGC proteins. n = 3 samples. m, In vivo quantification of nuclear Yap of heart sections from 12-week-old mice with PBS or agrin treatments one day after myocardial infarction. Representative images are shown. n = 1,167 cardiomyocytes from four mice per group. n, Cardiomyocyte proliferation assay of P7 heart cultures treated with agrin and the Yap–TEAD inhibitor. VP, verteporfin. n = 13,680 cardiomyocytes from eight samples. Scale bars, 10 μm (j) and 20 μm (m). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using a one-tailed t-test (b, c, j, m) or ANOVA with Dunnett’s post hoc test (f, g, n).

Article Snippet: Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Western Blot, Positive Control, Expressing, Membrane, Staining, Cell Cycle Assay, Immunofluorescence, Control, Fractionation, SDS Page, Recombinant, Isolation, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Proliferation Assay, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, b, Immunofluorescence of iPSC–CM cell-cycle activity by pH3 (a) or AURKB (b) in response to rat and human agrin administration (10–1,000 ng ml−1). Representative images of cultures treated with 1,000 ng ml−1 are shown. pH3 quantification was performed automatically using imageXpress software (n = 174,850 cardiomyocytes from 35 images of control (Ctrl) and 37 images of rat-agrin-treated cultures and 32 images for all other samples (a); n = 38,542 cardiomyocytes from nine control samples and six samples in all other groups (b). c, Human iPSC–CM proliferation assay in the 3D patch culture system. n = 3 samples for one week, 4 samples for other treatments. Scale bars, 20 μm (a–c). d–f, Effects of human agrin on conduction velocity (d) and structural and functional maturation genes (e, f). n = 4 samples (d); n = 3 samples per group (e, f). mRNA expression ratios of MYL2/MYL7 and TNNI3/TNNI1 are shown in e. CV, conduction velocity. n = 4 samples (d); n = 3 samples per group (e, f). Data are presented as mean ± s.e.m. proliferation per field. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a one-tailed t-test (d) or relative to untreated or 3-week control group using ANOVA followed by Dunnett’s post hoc test.

Article Snippet: Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Activity Assay, Software, Control, Proliferation Assay, Functional Assay, Expressing, One-tailed Test

Journal: Science translational medicine

Article Title: Advances in therapeutic applications of extracellular vesicles

doi: 10.1126/scitranslmed.aav8521

Figure Lengend Snippet: Recent disease treatment and tissue regeneration with EVs derived from MSCs. BM, bone marrow; ESC-MSCs, embryonic stem cell–derived MSCs; hiPSCs, human induced pluripotent stem cells; IL-10, interleukin-10; NK, natural killer; PEG, polyethylene glycol; SEC, size exclusion chromatography; TFF, tangential flow filtration; TNF-α, tumor necrosis factor–α; VEGF, vascular endothelial growth factor; UC, ultracentrifugation.

Article Snippet: Cardiovascular Myocardial infarction (MI) Rat BM MSCs Total exosome isolation reagent (Invitrogen) MSC-EVs reduced apoptosis and the myocardial infarct size and up-regulated myocardial LC3B expression as well as improved heart function in rat models of myocardial ischemia reperfusion injury ( 45 ).

Techniques: Derivative Assay, Size-exclusion Chromatography, Filtration, Isolation, Centrifugation, In Vitro, In Vivo, Functional Assay, Expressing, Transmission Assay, Mouse Assay, Chromatography

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: Collection of 35 zebrafish insertional cardiac (ZIC) mutants.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques:

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: Shown are immunostaining of a whole mice heart section using an anti-DNAJB6 antibody. Scale bar: 50 μm. RA, rigt atrium. RV, right ventricle. LA, left atrium. LV, left ventricle.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Immunostaining

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) The anti-DNAJB6 antibody immunostaining signal largely overlapped with the HCN4 immunostaining signal in the mouse SAN tissues under low magnification. ( B ) Under higher magnification, expression of DNAJB6 (red) only partially overlapped with HCN4 (green) as revealed by antibody co-immunostaining. Arrows point to cells with overlapping patterns. Stars indicate cells with no-overlapping. ( C ) Shown are fluorescent images after DNAJB6 and TBX3 antibody co-immunostaining indicating expression of DNAJB6 protein in the WT versus Dnajb6 +/- +/- mouse SAN. Arrows point to cells with weak DNAJB6 but strong TBX3 immunostaining signal. Stars indicate cells with strong DNAJB6 but low level of TBX3 immunostaining signal. ( D ) Quantification and correlation analysis of DNAJB6 and TBX3 immunostaining signal in WT SAN. ( E ) Quantification analysis of TBX3 signal in the WT versus Dnajb6 +/- mouse SAN. N=20 cells. Unpaired student’s t -test. Scale bars in A, 50 µm; In B, C, D, 20 µm.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Immunostaining, Expressing

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) Schematics of the Dnajb6 knockout (KO) mice. The insertion of Velocigene cassette ZEN-Ub1 created a deletion of 36,843 bp nucleotides spanning from the first to the last intron of the Dnajb6 gene at the Chromosome 5. The neomycin selection cassette was excised after crossed to a Cre expression line. ( B ) Representative DNA gel images of PCR genotyping for identifying WT (300 bp), Dnajb6 +/- heterozygous (hets), and Dnajb6 -/- homozygous (homo) mutant alleles . ( C ) Western blotting and quantification of DNAJB6 short (S) and long (L) protein expression in WT and Dnajb6 mutants. N=3 animal per group. ( D ) Shown are representative ECG recordings results showing SA and AVB phenotypes detected in the Dnajb6 +/- mice at 6 months. ( E ) The Dnajb6 +/- mice manifests impaired response to different autonomic stimuli. N=10–12 mice per group, unpaired student’s t -test. SA, sinus arrest. AVB, atrioventricular block. Figure 4—source data 1. Uncropped DNA gel image of PCR genotyping for identifying WT and DNAJB6 mutant mouse alleles (in PPT format). Figure 4—source data 2. Uncropped Western blot to show expression levels of DNAJB6 short (S) and long (L) proteins in WT and DNAJB6 mutants (in JPG format). Figure 4—source data 3. Uncropped Western blot to show expression levels of DNAJB6 short (S) and long (L) proteins in WT and DNAJB6 mutants (in PPT format). Figure 4—source data 4. Uncropped Western blot to show expression levels of DNAJB6 short (S) and long (L) proteins in WT and DNAJB6 mutants (in JPG format).

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Knock-Out, Selection, Expressing, Mutagenesis, Western Blot, Blocking Assay

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: Echocardiography indices in the Dnajb6 +/- mice compared to WT controls at 1 year.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques:

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) Heart morphology of Dnajb6 +/- mice compared to WT control at 1 year of age. ( B ) Quantification analysis of heart weight normalized by body weight (HW/BW) of Dnajb6 +/- mice compared to WT control at 1 year of age. N=4, unpaired student t test. ( C–E ) Shown are histology images of H&E staining ( C ), Masson’s Trichrome staining ( D ), and transmission electron microscope ( E ) indicating the myocardium structure of Dnajb6 +/- mice appeared to be indistinguishable to that in WT control. Scale bars in A, 1 mm; In C, D, 100 µm; In E, 5 um.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Staining, Transmission Assay, Microscopy

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ECG quantification of Dnajb6 heterozygous mice at 6 months of age.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques:

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) Leading pacemakers were located and plotted from both WT (blue dots) and Dnajb6 +/- (red dots) mice. One mouse could have multiple leading pacemaker locations due to the competing pacemakers and ectopic activities. SVC and IVC, superior and inferior vena cava; RAA, right atrial appendage; CT, crista terminalis; IAS, inter-atrial septum; AVN, atrioventricular node. Distribution of the leading pacemakers is summarized in panel. ( B ) Majority of leading pacemakers located within the SAN area in WT, whereas, in Dnajb6 +/- mice, significant increase of leading pacemakers locating in subsidiary pacemaker area and IAS was observed. p-value by Fisher exact test. ( C–D ) Activation map based on the optical mapping of action potentials showed representative leading pacemaker locations in WT (SAN) and Dnajb6 +/- mice (SAN and IAS areas). ( E ) Optical mapping on isolated atrial preparation showed bradycardia (baseline) and different responses of heart rate during isoproterenol, atropine, and carbachol stimulations between WT and Dnajb6 +/- mice. N=7–9 mice per group. Unpaired student’s t -test. ( F ) Increased cycle length (CL) variation was observed in Dnajb6 +/- isolated atrial preparations during different autonomic stimulations. N = 7–9 mice per group, unpaired student’s t -test.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Activation Assay, Isolation

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A–B ) Representative activation maps reconstructed for the last pacing stimulus ( S1 ) and the first spontaneous post-pacing atrial beat ( A1 ) during SAN recovery time (SANRT) measurements are shown. A site of the earliest atrial activation is labeled by a white asterisk. In Dnajb6 +/- group, unlike WT, the first spontaneous post-pacing atrial beat ( A1 ) originated from an ectopic location outside of the anatomically and functionally defined SAN area. ( C ) Summarized data for corrected SANRT (+/-) measured during different autonomic stimulations is shown. N=7–9 mice per group, unpaired student’s t -test.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Activation Assay, Labeling

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) Time course of membrane potential (E m ) predicted simulating our computational model of mouse SAN myocyte before (baseline) and after administration of isoproterenol (ISO) or carbachol (CCh). ( B ) Histogram illustrating the effects of ISO and CCh administration on firing rate (HR) distribution in our population of models. ( C ) Scatter plot quantifying HR variation in each model in the population. Red dots correspond to model variants resembling properties observed in ex vivo Dnajb6 +/ - mouse experiments ( +/- slower baseline HR, enhanced response to ISO, and reduced response to CCh), while the remaining model variants in grey mimic WT mouse functional measurements. ( D ) Histograms comparing the distribution of baseline HR in the two subgroups, and bar graphs reporting average ( ± SD) baseline HR, and relative HR variation after ISO and CCh administration in the two subgroups. ( E ) Bar graph reporting the differences between average model parameters’ scaling factors in the two subgroups. Note that a positive (negative) bar corresponds to increased (decreased) average parameter value in Dnajb6 +/- vs. WT groups. Asterisks in panels D and E indicate significant difference according to the 2-sided Wilcoxon rank sum test (performed with the MATALB function ranksum ). ( F ) Statistical analysis on the values of scaling factors of selected model parameters (G CaL , v NCX , v RyR , and G K,ACh ) performed with the MATLAB command boxplot . The central line indicates the median of each group ( q 50 ). The central box represents the central +/- % of the data, with lower and upper boundaries corresponding, respectively, to the 25 th and 75 th percentiles ( q 25 and q 75 ). The dotted vertical lines extend to 1.5 times the height of box, and individual values falling outside this range (shown here with grey circles) are considered outliers. The extremes of the lateral notches of the central box (determined as q 50 ± 1.57(q 75 –q 25 )/sqrt( n ), where n is the number of observations in each group) mark the 95% confidence interval for the medians. When the notches from two boxplots do not overlap, as in the four cases shown here, one can assume that the medians are different with a significance level of 0.05.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Ex Vivo, Functional Assay

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) Principal component analysis (PCA) reveals the variance in transcriptome distribution in the Dnajb6 +/- mice and WT controls at 6 months. Each point represents the projections of individual hearts onto principal component (PC). ( B ) Heatmap of genes differentially expressed in the Dnajb6 +/- mice and WT controls. Each column represents an individual replicate and there are 3 replicates per group. Each row represents an individual gene. The color bar represents relative expression of log-transformed, normalized counts with upregulated genes shown in red and downregulated genes in blue. ( C ) Volcano plot shows magnitude and significance of genes that altered in distribution in the Dnajb6 +/- mice and WT controls. Genes that significantly downregulated in the Dnajb6 +/- mice are plotted in blue (left) and upregulated are plotted in red (right). ( D ) Ingenuity Pathway Analysis (IPA) of differentially expressed genes in the Dnajb6 +/- mice and WT controls. Signaling pathways are organized in the order of significance as –log10 of P value.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Expressing, Transformation Assay

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: ( A ) Expression of six calcium homeostasis and ion channel related genes were altered in the Dnajb6 +/- mice right atrium. ( B ) Expression of four Wnt pathway related genes were altered in the Dnajb6 +/- mice right atrium. ( C ) Quantitative polymerase chain reaction (qPCR) validation of DE genes listed in A and B, normalized to Gapdh; RNA was extracted from an individual moue right atrium, which was considered a single biological replicate. Samples were collected in triplicate. N=3 mice per group, unpaired student’s t -test. Figure 7—source data 1. A list of 107 differentially expressed genes identified between DNAJB6+/- knockout and WT mouse.

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Knock-Out

Journal: eLife

Article Title: A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

doi: 10.7554/eLife.77327

Figure Lengend Snippet: Identification of the DNAJB6 variant from patients with sick sinus syndrome (n=162).

Article Snippet: The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302).

Techniques: Variant Assay

Journal: Nature Communications

Article Title: A cross-study transcriptional patient map of heart failure defines conserved multicellular coordination in cardiac remodeling

doi: 10.1038/s41467-025-62219-6

Figure Lengend Snippet: A Similarities of the molecular descriptions of HF between pairs of bulk or single-nucleus (SN) transcriptomics studies were approximated with the performance of disease classifiers built with each study of our core curation. The disease classifier from bulk studies used the changes in expression of each gene, while the classifiers of SN-studies were built with cell-type compositions or multicellular programs. B Area under the receiver operating characteristic curve (AUROC) of pairwise predictions of disease classifiers built from all individual bulk studies included in our core collection using the top 500 differentially expressed genes. Red labels mark the study expansion. C Gene ranking of conservation of gene deregulation events in HF across bulk transcriptomics studies based on the adjusted p value of a Fisher combined analysis. The dotted line shows the top-500 genes. D Hierarchical clustering of all SN-transcriptomics tissue samples based on the composition of the seven major cell-types used in our ontology. E T-statistics (heatmap) and estimate of difference between failing and non-failing hearts from the differential compositional analysis using t-tests and linear mixed models, respectively. Stars denote an adj. p value < 0.05. F AUROC of pairwise predictions from disease classifiers built from all core SN studies using cell-type compositions. G Uniform Manifold Approximation and Projections (UMAP) of the multicellular programs describing the variability of the respective tissue samples of each core SN study. Tissue samples of the rest of the studies are projected into each latent space and distinguished with the shape of the dot. Colors highlight different HF etiologies. All plots include 132 patients. H AUROC of pairwise predictions from disease classifiers built from all core SN-studies using multicellular programs. In all panels Cardiomyocytes (CM), fibroblasts (Fib), pericytes, endothelial (Endo), and vascular smooth muscle (vSMCs) cells. Heart failure (HF), and non-failing (NF) hearts. Source data are provided as a Source Data file.

Article Snippet: Neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from 1- to 2-day-old Sprague-Dawley rat pups using a modified protocol adapted from Miltenyi Biotec’s Neonatal Heart Dissociation Kit (mouse/rat, 130-098-373) and Neonatal Cardiomyocyte Isolation Kit (rat, 130-105-420).

Techniques: Expressing

Journal: Nature Communications

Article Title: A cross-study transcriptional patient map of heart failure defines conserved multicellular coordination in cardiac remodeling

doi: 10.1038/s41467-025-62219-6

Figure Lengend Snippet: A Multicellular factor analysis was used to integrate the patients' profiles across the single-nucleus core studies. The integrative model represents each sample in terms of latent variables, referred here as multicellular programs (MCP), that capture gene expression variability across cell types, patients, and studies. Each MCP can be understood as a collection of genes whose expression is coordinated across cell types. B Patient map built from MCP1 and MCP2 with samples ( n = 132) colored by their disease status, etiology, and study of origin. C Mean standardized gene expression of the top 5 genes in MCP1 whose expression was captured in all studies. Data were grouped by disease status and study of origin. D Functional dissection of the patient map built from MCP1 and MCP2 from a multicellular (left) or cell-type (right) perspective. Each functional vector represents the level of enrichment of a function in the location of the map where the arrow points to. The larger the arrow, the more enriched the function. Gene sets were manually selected for representation from a set of gene sets enriched in either MCP1 and/or MCP2 (adj. two-sided p value < 0.1, hypergeometric test). E Multicellular coordination network of HF processes captured by MCP1, where each arrow describes how important the expression of a given cell-type is to predict the expression profile of another one (Methods). Predictive importances come from linear mixed models of cell-type signatures of MCP1. Importances below 0.2 were not included. F Association between the predictive importance of a pair of cell-types (sender and target) and the number of potential ligand-receptor coexpression events. Pairs of cells are colored by their target cell-type and highlighted when fibroblasts (Fibs) are the sender cell-type. Pearson’s correlation coefficient and its p value is displayed. G Regulatory potential score, as estimated by NicheNet, represents the potential of fibroblasts’ ligands in contributing to the regulation of cardiomyocyte genes (MCP1 gene loading < −0.2). In all panels, Cardiomyocytes (CM), fibroblasts (Fib), pericytes (PC), endothelial (Endo), vascular smooth muscle (vSMCs) cells. Heart failure (HF), and non-failing (NF) hearts. Source data are provided as a Source Data file.

Article Snippet: Neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from 1- to 2-day-old Sprague-Dawley rat pups using a modified protocol adapted from Miltenyi Biotec’s Neonatal Heart Dissociation Kit (mouse/rat, 130-098-373) and Neonatal Cardiomyocyte Isolation Kit (rat, 130-105-420).

Techniques: Gene Expression, Expressing, Functional Assay, Dissection, Plasmid Preparation

Journal: Nature Communications

Article Title: A cross-study transcriptional patient map of heart failure defines conserved multicellular coordination in cardiac remodeling

doi: 10.1038/s41467-025-62219-6

Figure Lengend Snippet: A Cell-type specific transcriptional processes associated with MCP1 and MCP2 are enriched in every patient tissue sample from the bulk core study collection ( n = 1392). Upper panels show AUROC distributions ( n = 21) evaluating how well cell-type-specific responses classify non-failing hearts in each study. B Schematic of potential tissue-level regulatory mechanisms underlying observed gene expression changes in bulk data. Upregulation in HF may reflect increased abundance of cell types expressing the gene (Compositional regulation, Comp.), increased gene expression within cells (Molecular, Mol.), or both (Comp/Mol). Downregulation follows similar principles. C Consensus gene-level statistics of HF-associated expression changes from bulk ( x axis) and compositional (upper) or molecular (lower) regulation from single-nucleus data ( y axis). Genes shown are from the top 8942 in the consensus bulk ranking (adj. Fisher p value < 0.05) (Fig. ). D Annotation of deregulation events derived from the combination of bulk and single-nucleus transcriptomics studies. The annotation comes from the top 8942 genes of the consensus ranking (adj. two-sided Fisher p value < 0.05). E Root mean square error and Pearson correlation of pseudobulk deconvolution results (core and supporting single-nucleus data). Each dot is one dataset, stratified by tissue type (HF, n = 10; NF, n = 8) and signature gene set used ( x axis). Unreg, unregulated; dereg, deregulated; dereg mol, molecularly deregulated; dereg comp, compositionally deregulated. Two-sided paired Wilcoxon test. F Hierarchical clustering of bulk RNA-seq samples (N = 697) based on deconvoluted composition using a healthy reference (subset to compositionally regulated genes and seven major cell types). G Heatmap of t -statistics and estimated differences between HF and NF hearts from differential compositional analysis using t tests and linear mixed models. Stars: adj. two-sided p value < 0.05. In all panels cardiomyocytes (CM), fibroblasts (Fib), pericytes (PC), endothelial (Endo), vascular smooth muscle (vSMCs) cells. Heart failure (HF), and non-failing (NF) hearts. Boxplots in ( A , E ) display the minimum, first quartile (Q1), median, third quartile (Q3), and maximum; outliers lie beyond 1.5 times the interquartile range (IQR) from Q1 or Q3 and are shown as points. Source data are provided as a Source Data file.

Article Snippet: Neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from 1- to 2-day-old Sprague-Dawley rat pups using a modified protocol adapted from Miltenyi Biotec’s Neonatal Heart Dissociation Kit (mouse/rat, 130-098-373) and Neonatal Cardiomyocyte Isolation Kit (rat, 130-105-420).

Techniques: Gene Expression, Expressing, Derivative Assay, RNA Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: Subcellular progression of mesenchymal transition identified by two discrete synchronous cell lines derived from the same glioblastoma

doi: 10.1007/s00018-022-04188-3

Figure Lengend Snippet: RNA-seq analysis. a Pearson correlation of the overall expression profile showing minimal differences between subclone #5 and #11 with the parent tissue and cells. The numbers in the boxes represent the correlation coefficient. Each subclone has expression correlated 78% and 98% with the parent tissue and cells, respectively. The correlation between the two subclones was 92%. b Volcano plot for identifying differentially expressed genes in subclone #5 and #11 showing significant amounts of DEGs between the two subclones despite similar overall gene expression. c Heatmap of the top 20 differentially expressed genes between subclone #5 (group 2) and #11 (group 1). d Functional annotations of significantly upregulated genes in subclone #11 compared to subclone#5 (FC ≥ 2, P value < 0.05) showing the EMT and cell cycle-related gene ontology clusters being significantly upregulated in subclone #11 (ES > 1.5, P value < 0.05). e Western blot analysis for EMT-related protein expression in SNU-4210, subclone #5, and #11 showing higher expression of E-cadherin, vimentin and slug in subclone #11 compared to #5

Article Snippet: Antibodies against TGF-β (3711), Vimentin (5741), N-Cadherin (13,116), Claudin-1 (13,255), β-Catenin (8480), ZO-1 (8193), Snail (3879), Slug (9585), TCF/ZEB1 (3396), E-cadherin (3195), phosphorylated Smad2 at Ser465/467 (3108), Smad2 (5339), phosphorylated Smad3 at Ser423/425 (9520), Smad3 (9523), Smad4 (38,454) and ACTB (4967) were from Cell Signalling Technology; and HRP-conjugated IgGs (111-035-003 and 115-035-003) were from Jackson Immune Research.

Techniques: RNA Sequencing, Expressing, Gene Expression, Functional Assay, Western Blot