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: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

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: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

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: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

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: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

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: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

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

Journal: iScience

Article Title: The prion protein is required for normal responses to light stimuli by photoreceptors and bipolar cells.

doi: 10.1016/j.isci.2024.110954

Figure Lengend Snippet: Figure 7. Morphometric analysis of ribbon synapses and cone pedicle anatomy (A and B) Confocal maximum intensity projections show representative retinal sections from 129/Ola WT and PrPKO mice. PKCa (green) marks cell bodies and dendrites of rod bipolar cells, CtBP2 (yellow) marks synaptic ribbons and Pikachurin (magenta) is a protein present in the cleft of ribbon synapses. (C and D) 3D surfaces were rendered using IMARIS software 9.9.1. to enable quantitation of ribbons and synaptic puncta, assessment of 3D morphology, and relative associations of synaptic ribbons, Pikachurin and rod-bipolar cell dendrites. (E and F) Quantitation of ribbons (CtBP2), synaptic cleft puncta (Pikachurin) and ratio of ribbons to synaptic puncta are shown in graphs for both 129/Ola and C57BL/6J retinas. (G and H) Cone pedicle (green) and ribbon synapse (yellow) 3D surfaces were rendered using IMARIS software 9.9.1. For 129/Ola -WT and PrPKO, top panels show pre-rendered confocal image and bottom panels show 3D rendered images. Example cone pedicles with volume calculation are shown at right. Each cone pedicle was rotated in 3D to study associations with ribbon synapses (yellow). (I and J) Quantitative data from analysis of confocal datasets in E, are shown for cone pedicle volumes, and the number of ribbon synapses per cone pedicle. Scale bars: A, B = 4 mm (left), 2 mm (right) C, D = 1 mm G, H = 5 mm (left), 1 mm (right). Statistical analysis by Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, bars represent mean values, +/ SD.

Article Snippet: Antibodies D13-anti prion protein (Human) 1:100 Matsuanaga66 N/A Anti-Cone Arrestin - Arrestin 3/Cone photoreceptors – Rabbit - 1:100 Millipore AB15282; RRID:AB_1163387 Anti-CtBP2 - C-terminal binding protein 2/ribeye protein of ribbon synapses - Mouse 1:100 Santa Cruz sc-17759; RRID:AB_627310 Anti-PKCa - Protein kinase C/Rod bipolar cells - Goat 1:100 RD Systems AF5340; RRID:AB_2168552 Anti -Pikachurin (ribbon synaptic cleft) - Rabbit 1:100 Proteintech 14578-1-AP; RRID:AB_2277653 Anti- EAAT1 - Excitatory amino acid transporter - Rabbit 1 1:100 Abcam ab416; RRID:AB_304334 Anti-EAAT5 - Excitatory amino acid transporter 5 - Rabbit 1:100 Abcam ab230217 Anti -PSD-95 - Post synaptic density-95 - Mouse 1:100 Neuromics MO50000 Anti-Calbindin – horizontal cells – Rabbit 1:100 Abcam Ab108404; RRID:AB_10861236 Critical commercial assays Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 10X Genomics v3.1

Techniques: Software, Quantitation Assay, MANN-WHITNEY

Journal: Cancer research

Article Title: Cancer-cell-derived matrisome proteins promote metastasis in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-19-2578

Figure Lengend Snippet: A, a proteomic pipeline was used to identify overrepresented matrisome proteins in PDAC, and these proteins were then assigned as originating from human (cancer cell) or mouse (stroma) by MS analyses of human-to-mouse xenograft tumor ECM (13). Analyses of correlations with patient survival of individual matrisome proteins of different origins identified cancer-cell-derived matrisome proteins as being correlated with poor patient overall survival (e.g. AGRN, SERPINB5, and CSTB) whereas stromal-cell-derived proteins correlated either with good or poor survival and many showed no correlation with survival.

Article Snippet: Immunoblotting of tumor samples, cells and cell culture media Tumor samples were lysed in Laemmli buffer, proteins were separated by SDS-PAGE (4–20% gradient gel from Biorad) and immunoblotting was performed using the following antibodies: GAPDH (Millipore, Bedford, MA, MAB374), AGRN (Novus Biologicals, Littleton, CO, NBP1–90209), SERPINB5 (Origene, Rockville, MD, TA-322980), CSTB (Abcam, Cambridge, MA, ab53725).

Techniques: Derivative Assay

Journal: Cancer research

Article Title: Cancer-cell-derived matrisome proteins promote metastasis in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-19-2578

Figure Lengend Snippet: A-F, double-color IHC with human-specific LMNA in brown and ZEB1 (A,D), VIM (B,E), or CDH1 (C,F) in red. The corresponding quantifications (see Materials and Methods) show that AGRN kd, but not SERPINB5 or CSTB kd, reduced ZEB1+ and VIM+ tumor-cell fractions and increased the CDH1+ tumor-cell fraction, while AGRN oe regulated EMT in the opposite direction.

Article Snippet: Immunoblotting of tumor samples, cells and cell culture media Tumor samples were lysed in Laemmli buffer, proteins were separated by SDS-PAGE (4–20% gradient gel from Biorad) and immunoblotting was performed using the following antibodies: GAPDH (Millipore, Bedford, MA, MAB374), AGRN (Novus Biologicals, Littleton, CO, NBP1–90209), SERPINB5 (Origene, Rockville, MD, TA-322980), CSTB (Abcam, Cambridge, MA, ab53725).

Techniques:

Journal: Cancer research

Article Title: Cancer-cell-derived matrisome proteins promote metastasis in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-19-2578

Figure Lengend Snippet: A, CRISPR-inactivation (kd) knocked down expression in the BxPC3 G1.1 cell line, of AGRN, SERPINB5 and CSTB, respectively, as shown by qPCR (top) and western blot (WB) (bottom). GAPDH is the loading control.

Article Snippet: Immunoblotting of tumor samples, cells and cell culture media Tumor samples were lysed in Laemmli buffer, proteins were separated by SDS-PAGE (4–20% gradient gel from Biorad) and immunoblotting was performed using the following antibodies: GAPDH (Millipore, Bedford, MA, MAB374), AGRN (Novus Biologicals, Littleton, CO, NBP1–90209), SERPINB5 (Origene, Rockville, MD, TA-322980), CSTB (Abcam, Cambridge, MA, ab53725).

Techniques: CRISPR, Expressing, Western Blot

Journal: Cancer research

Article Title: Cancer-cell-derived matrisome proteins promote metastasis in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-19-2578

Figure Lengend Snippet: A, CRISPR-SAM-induced overexpression (oe) in the AsPC1 cell line, overexpressed AGRN, SERPINB5 and CSTB, respectively, as shown by qPCR (top) and western blot (WB) (bottom). GAPDH is the loading control.

Article Snippet: Immunoblotting of tumor samples, cells and cell culture media Tumor samples were lysed in Laemmli buffer, proteins were separated by SDS-PAGE (4–20% gradient gel from Biorad) and immunoblotting was performed using the following antibodies: GAPDH (Millipore, Bedford, MA, MAB374), AGRN (Novus Biologicals, Littleton, CO, NBP1–90209), SERPINB5 (Origene, Rockville, MD, TA-322980), CSTB (Abcam, Cambridge, MA, ab53725).

Techniques: CRISPR, Over Expression, Western Blot