Journal: bioRxiv

Article Title: Role of PARP1 in oligodendrocyte differentiation during developmental myelination and remyelination after myelin damage

doi: 10.1101/2021.04.23.441060

Figure Lengend Snippet: A, flow chart of identifying PARylated acceptor proteins. Whole proteins were prepared from the spinal cord of P7 mice 30 min after PARG inhibitor PDD injection to stabilize protein PARylation (PAR half-life is less than 1min). B-C , GO analysis of biological processes ( B ) and molecular function ( C ) of the 131 potential PARylated acceptor proteins identified by Co- IP and LC-MS/MS. D, representative graphs depicting examples of known PARylated proteins and novel PARylated proteins. E, Co-IP followed by Western blotting assay confirming the interaction between Myef2 and PARP1 and the presence of PARylated Myef2 at the molecular weight of Myef2 (∼55kD) in the primary brain OLs at D2. Rabbit IgG, negative control for Myef2 IP. F, Myef2 mRNA level in primary OPCs and D1, 2 and 4 differentiating OLs. N = 3 each group. One-way ANOVA followed by Tukey’s multiple comparison tests. G, double IHC of PARP1 and Myef2 in the corpus callosum of P7 Parp1-WT and Parp1-KO mice. H , abundance of Mbp and Mag mRNA in the immunoprecipitates using RNA immunoprecipitation (RIP) by Myef2 antibody or Ctrl IgG in D2 primary OLs treated with DMSO or 4HQ treated. N = 3 each group. Two-way ANOVA followed by Sidak multiple comparison tests. Mbp: DMSO vs 4HQ F (1, 8) = 10.79 P = 0.0111, IgG vs Myef2 antibody F (1, 8) = 53.51 P < 0.0001. Mag: DMSO vs 4HQ F (1, 8) = 10.82 P = 0.0110, IgG vs Myef2 antibody F (1, 8) = 43.75 P = 0.0002. I , schematic conclusion of panel H , PARP1 inhibition by 4HQ potentiated the binding of Myef2 to mRNA molecules. J , experimental designs for Panels K-P . Primary OPCs purified from neonatal rat brain were differentiated in the presence of Myef2 siRNA or scramble controls for 36 h in the differentiation medium prior to analysis. K, qRT-PCR assay for Myef2 mRNA level. N = 4 Ctrl, 4 Myef2 siRNA. Myef2, t (6) = 21.36 P <0.0001. L , ICC of Myef2 and MBP in Ctrl and Myef2 siRNA-treated primary OLs. M , quantification of Myef2 intensity and MBP + ramified OLs (percentage of DAPI + cells). N = 5 Ctrl, 4 Myef2 siRNA. Myef2 intensity , t (8) = 6.084 P = 0.0003; % of MBP + cells, t (8) = 6.672 P = 0.0002. N-O, representative Western blotting images ( N ) and quantification ( O ). β-actin, loading control. N = 3 scramble Ctrl, 4 Myef2 siRNA. Myef2 , t (4) = 3.410 P = 0.0270; MAG, t (4) = 2.893 P = 0.0444; MBP, t (4) = 3.387 P = 0.0276. P, qRT-PCR assay for Mbp and Mag in Ctrl (n = 4) and Myef2 siRNA-treated (n = 4) primary OLs. Mbp, t (6) = 0.2572 P = 0.8056; Mag , t (6) = 0.1497 P = 0.8859. Two-tailed Student’s t test, * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Scale bars: G, 20 μm; L 10 μm.

Article Snippet: Primary antibodies and isotype control IgG used were: PARP1 (39559, RRID: AB_2793257, Active Motif), PAR (4335-MC-100, RRID: AB_2572318, Trevigen), Sox2 (sc-17320, RRID: AB_2286684 Santa Cruz Biotechnology), Myef2 (16051-1-AP, RRID: AB_2146869, Proteintech), Normal rabbit IgG (#2729, RRID: AB_1031062, Cell Signaling Technology), Normal goat IgG (AB-108-C, RRID: AB_354267, R&D Systems).

Techniques: Injection, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Molecular Weight, Negative Control, Comparison, RNA Immunoprecipitation, Inhibition, Binding Assay, Purification, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Scientific reports

Article Title: Zebrafish VCAP1X2 regulates cardiac contractility and proliferation of cardiomyocytes and epicardial cells.

doi: 10.1038/s41598-018-26110-3

Figure Lengend Snippet: Figure 2. VCAP1X2 mutant displayed cardiac dysfunction and impaired cardiomyocyte proliferation. (A) Pseudodynamic reconstructed 3D images and respective bright-field images are shown of end-diastolic phase beating hearts from Tg(myl7:EGFP; myl7:H2AFZ mCherry) (a,b) or VCAP1X2 mutant (c,d) at 72 hpf. Yellow lines indicate the diameter of the ventricular chamber. Green fluorescence marks ventricular myocardium and red indicates ventricular chamber. Scale bar, 50 μm. (B) Parameters related to heart performance were measured from pseudodynamic reconstructed 3D images (a–c, n = 3, N = 2) or CCD images (d–f, n = 20, N = 3) for Tg(myl7:EGFP; myl7:H2AFZ mCherry) or WT and VCAP1X2 mutant at 72 hpf. End-systolic volume (a) and end-diastolic volume (b) were significantly increased in VCAP1X2 mutant compared with Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos. Ejection fraction (c) was lower in VCAP1X2 mutant than in Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos. End-diastolic length (d) and width (e) were increased while fractional shortening (f) was decreased in VCAP1X2 mutant compared to WT. Error bars represent standard error. Student’s t-test, **p < 0.01, ***p < 0.001. (C) Paraffin sectioning with hematoxylin and eosin staining revealed a thinner compact layer in the ventricle of VCAP1X2 mutants (b) and LacZ mRNA-injected VCAP1X2 mutants (c) compared to WT (a) or VCAP1X2 mRNA-injected VCAP1X2 mutants (d) (n = 10 per condition). a, atrium, v, ventricle. Scale bar, 50 μm. (D) Immunostaining of BrdU (red) in heart ventricles from Tg(myl7:EGFP; myl7:H2AFZ mCherry) (myl7) (a) or VCAP1X2 mutants (b) at 72 hpf. Significantly decreased cardiomyocyte (CM) number (c) and percentage of BrdU+ CMs (d) in the ventricle was detected in VCAP1X2 mutant compared to Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos (n = 20 per condition, N = 3). Student’s t-test, ***p < 0.001. Scale bar, 50 μm. Apoptotic cells were analyzed by TUNEL labeling (E) and similar low level of apoptotic cells (F) was detected in VCAP1X2 mutant and Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos (n = 15, N = 3). Cardiomyocyte nuclei stained by anti-MEF2 antibody. Error bars represent standard error. Scale bar, 50 μm.

Article Snippet: Tissues were incubated with primary antibody including MEF2 (1:150, Santa Cruz Biotechnology) or PCNA (1:200, Abcam) at 4 °C overnight.

Techniques: Mutagenesis, Fluorescence, Staining, Injection, Immunostaining, TUNEL Assay, Labeling