Journal: International journal of biological sciences

Article Title: Heme-binding protein 1 delivered via pericyte-derived extracellular vesicles improves neurovascular regeneration in a mouse model of cavernous nerve injury.

doi: 10.7150/ijbs.81809

Figure Lengend Snippet: Figure 2. Hebp1 protein improves erectile function in CNI-induced ED mice. (A) Representative ICP responses for sham-operated and CNI-induced ED mice stimulated 1 week after two intracavernous injections (administered on days -3 and 0) of Hebp1 protein (1.0 and 5 µg per mouse). The stimulus interval is indicated by a solid bar. (B, C) Ratios of mean maximal ICP and total ICP (area under the curve) to MSBP were calculated for each group. Results are presented as means ± SEM (n = 5). (D, E) Immunofluorescence staining of CC and DNB tissues for PECAM-1 (D; green), NG2 (D; red), and NF (E; green) after ICP studies. Nuclei were labeled with the DNA dye DAPI (blue). Scale bars, 100 µm (D) and 25 µm (E). (F) NF (green) immunofluorescence staining in mouse MPGs exposed to LPS (2.5 μg/ml) and then treated with Hebp1 (5 µg/ml) or PBS for 5 days. Scale bar, 100 μm. (G–I) Quantitative analysis of CC endothelial cell (G, PECAM-1), pericyte (H, NG2) and neuronal cell (I, NF) contents using an image analyzer. Results are presented as means ± SEM (n = 5). (J) Neurite sprouting, quantified using an image analyzer. Results are presented as means ± SEM (n = 4; *P < 0.05, **P < 0.01, ***P < 0.001). The relative ratio of the sham operation or control group was defined as 1. ED, erectile dysfunction; CNI, cavernous nerve injury; NF, neurofilament; DAPI, 4,6-diamidino-2-phenylindole; MPG, major pelvic ganglion; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; MSBP, mean systolic blood pressure; DNB, dorsal nerve bundles; ns, not significant.

Article Snippet: Frozen tissue sections (15 μm) or ex vivo MPG samples were incubated with primary antibodies against the following proteins: Hebp1 (Ca# NBP2-14977, 1:100; NOVUS Biologicals), Hebp1 (Ca# sc-398750, 1:100; Santa Cruz Biotechnology Inc., Dallas, TX, USA), platelet/endothelial adhesion molecule 1 (PECAM-1, Ca# MAB1398Z, 1:100; Millipore), neuron-glial antigen 2 (NG2, Ca# AB5320, 1:100; Millipore), neurofilament (Ca# N5389, 1:50; Sigma-Aldrich), PDGFRβ (Ca# sc-1627, 1:50; Santa Cruz Biotechnology Inc.), phospho-histone 3 (Ca# 06-570, 1:50; Millipore), phospho-eNOS (Ca# PA5-17917, 1:50; Invitrogen, Carlsbad, CA, USA), oxidized low-density lipoprotein (oxidized-LDL, Ca# ab 14519, 1:100; Abcam, Cambridge, UK), or nitrotyrosine (Ca# 06-284, 1:100; Millipore) at 4°C overnight.

Techniques: Immunofluorescence, Staining, Labeling, Control, Saline

Journal: International journal of biological sciences

Article Title: Heme-binding protein 1 delivered via pericyte-derived extracellular vesicles improves neurovascular regeneration in a mouse model of cavernous nerve injury.

doi: 10.7150/ijbs.81809

Figure Lengend Snippet: Figure 4. Hebp1 is delivered by MCP-EVs and contributes to nerve regeneration. (A) Representative transmission electron microscopy (TEM) phase images for detecting isolated MCP-EVs. Scale bar = 100 nm. (B) Representative Western blot for Hebp1, EV positive markers (CD63, CD9 and CD81) or EV negative marker (GM130) in MCPs and MCP-EVs. (C, D) Expression of Hebp1 in MCP-EVs infected with lentivirus expressing shHebp1 or shCon at three doses (1 × 103, 5 × 104, and 1 × 104 TU/ml culture medium) for at least 3 days. (E, F) Densitometric quantification of Hebp1 protein bands using an image analyzer. Results are presented as means ± SEM (n = 4). (G, H) Immunofluorescence staining for NF in mouse MPG tissues exposed to LPS (2.5 μg/ml) and other indicated conditions for 5 days (G). Lengths of NF-positive neurites in MPG tissues, quantified using an image analyzer (H). Results are presented as means ± SEM (n = 4). Scale bar, 100 µm. (I, J) Migration assays of mouse Schwann cells exposed to LPS (2.5 μg/ml) and other indicated conditions for 24 hours (I). Number of migrated cells in the red dotted rectangle, quantified using an image analyzer (J). Results are presented as means ± SEM (n = 4; **P < 0.01, ***P < 0.001). The relative ratio of the shCon (control) group was defined as 1. MCPs, mouse cavernous pericytes; EVs, extracellular vesicles; LPS, lipopolysaccharide; MPG, major pelvic ganglion; PBS, phosphate-buffered saline; ns, not significant.

Article Snippet: Frozen tissue sections (15 μm) or ex vivo MPG samples were incubated with primary antibodies against the following proteins: Hebp1 (Ca# NBP2-14977, 1:100; NOVUS Biologicals), Hebp1 (Ca# sc-398750, 1:100; Santa Cruz Biotechnology Inc., Dallas, TX, USA), platelet/endothelial adhesion molecule 1 (PECAM-1, Ca# MAB1398Z, 1:100; Millipore), neuron-glial antigen 2 (NG2, Ca# AB5320, 1:100; Millipore), neurofilament (Ca# N5389, 1:50; Sigma-Aldrich), PDGFRβ (Ca# sc-1627, 1:50; Santa Cruz Biotechnology Inc.), phospho-histone 3 (Ca# 06-570, 1:50; Millipore), phospho-eNOS (Ca# PA5-17917, 1:50; Invitrogen, Carlsbad, CA, USA), oxidized low-density lipoprotein (oxidized-LDL, Ca# ab 14519, 1:100; Abcam, Cambridge, UK), or nitrotyrosine (Ca# 06-284, 1:100; Millipore) at 4°C overnight.

Techniques: Transmission Assay, Electron Microscopy, Isolation, Western Blot, Marker, Expressing, Infection, Immunofluorescence, Staining, Migration, Control, Saline

Journal: Nucleic Acids Research

Article Title: Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

doi: 10.1093/nar/gkq1241

Figure Lengend Snippet: A substantial amount of damages are still being repaired 2 h after exposure to DMS. Level of SSBs in EM9-XH cells treated with ( A ) DMS (2 mM) or ( B ) mock treated and left to repair. PARP inhibitor was added (open symbols, time point indicated by arrow) to slow down the ligation step, 2 h after treatment was terminated. The means and standard errors of three experiments are shown. The ongoing SSB formation 2 h after terminated treatment, represents MPG initiated BER events. Level of SSBs in A549 cells with or without siRNA depleted MPG and treated with (A) DMS (0.5 mM) or (B) mock treated and left to repair. PARP inhibitor was added (open symbols, time point indicated by arrow) to slow down the ligation step, 2 h after treatment was terminated. The means and standard errors of three experiments are shown.

Article Snippet: The lysates (75 μg protein) were resolved by 4–12% NuPAGE Bis-Tris Gel (Invitrogen) for subsequent western blot analysis using antibodies against the following proteins diluted in 5 % milk (in PBS) as indicated: β-tubulin (1:500), PARP1 (1:500) and MPG (HPA006531, Atlas Antibodies AB) (1:500).

Techniques: Ligation

Journal: Nucleic Acids Research

Article Title: Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

doi: 10.1093/nar/gkq1241

Figure Lengend Snippet: The glycosylase MPG is required for the formation of the majority of SSBs produced during the repair of DMS-induced damages. ( A ) Western blot, probed with antibodies against MPG and loading control β-tubulin, showing the siRNA knockdown of MPG in human A549 cells. ( B ) Levels of SSBs in wild-type A549 cells and cells depleted of MPG, after a 15 min treatment with 0.5 mM DMS at indicated time points of repair. Cells were treated and left to repair in the absence or presence of PARP inhibitor. The means and standard errors of three experiments are shown.

Article Snippet: The lysates (75 μg protein) were resolved by 4–12% NuPAGE Bis-Tris Gel (Invitrogen) for subsequent western blot analysis using antibodies against the following proteins diluted in 5 % milk (in PBS) as indicated: β-tubulin (1:500), PARP1 (1:500) and MPG (HPA006531, Atlas Antibodies AB) (1:500).

Techniques: Produced, Western Blot, Control, Knockdown

Journal: Nucleic Acids Research

Article Title: Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

doi: 10.1093/nar/gkq1241

Figure Lengend Snippet: Model for BER and the influence of PARP. The MPG glycosylase is recognizing N-methylated purines, likely through scanning the DNA for base lesions. Once a lesion is recognized, MPG excises the damaged base and the APE1 endonuclease cleaves the newly formed AP-site into a SSB intermediate. If this intermediate is uncoupled from the repair process before repair is finished, PARP1 may recognize and bind to the SSB intermediate, which overall might slow down the BER process. In the event that PARP1 is inhibited it will be trapped on the SSB and prevent ligation. However, PARP1 is not required for accurate repair if the repair pathway remains intact. In the event of the BER complex being unable to accurately ligate, long patch repair is thought to take over. Our model does not exclude an active role for PARP1 in BER by influencing DNA damage-induced chromatin remodelling.

Article Snippet: The lysates (75 μg protein) were resolved by 4–12% NuPAGE Bis-Tris Gel (Invitrogen) for subsequent western blot analysis using antibodies against the following proteins diluted in 5 % milk (in PBS) as indicated: β-tubulin (1:500), PARP1 (1:500) and MPG (HPA006531, Atlas Antibodies AB) (1:500).

Techniques: Methylation, Ligation