Journal: DNA repair

Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

doi: 10.1016/j.dnarep.2019.02.001

Figure Lengend Snippet: (A) For each sample, linear DNA substrate containing a single uracil or an F residue was incubated with 50 μg of POLB−/− extract, and where indicated, supplemented with 0.5 pmol of Polβ, with 0.5 pmol of Polλ−39, or (C) with 0.5 pmol of full-length Polλ. Reactions were performed and analyzed as described in Section 2. The means ± standard errors are plotted (n=3). Data points from experiment POLB−/− extract (Fig.1) are also plotted for reference. (B) A schematic showing the domain organization of Polλ.

Article Snippet: Full-length human Polλ protein and rabbit anti-Polλ polyclonal antibody were kind gifts from Drs. Thomas Kunkel and Samuel H. Wilson respectively.

Techniques: Residue, Incubation

Journal: DNA repair

Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

doi: 10.1016/j.dnarep.2019.02.001

Figure Lengend Snippet: The linear uracil and F substrates were used in a series of experiments where the indicated amounts of purified Polβ (left) or Polλ (right) were added to POLB−/− extracts. Reactions were performed with an incubation time of 30 min at 37°C and analyzed as described in Section 2.5 and 2.6. The means ± standard errors are plotted (n=3).

Article Snippet: Full-length human Polλ protein and rabbit anti-Polλ polyclonal antibody were kind gifts from Drs. Thomas Kunkel and Samuel H. Wilson respectively.

Techniques: Purification, Incubation

Journal: DNA repair

Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

doi: 10.1016/j.dnarep.2019.02.001

Figure Lengend Snippet: (A) Domain organization of Polβ showing the position of residues changed in the Polβ variants. (B) Repair of uracil and F-containing linear substrates in WT, POLB−/− MEF extracts and extracts from POLB−/− cells expressing lyase-dead (K35A/K68A/K72A) or polymerase-dead (D256A) variants of Polβ, with Polλ, or with Polλ−39. Data points from experiment with WT and POLB−/− extract (Fig.​(Fig.1)1) are also plotted for reference. (C) Repair of uracil and F-containing linear substrates in POLB−/− MEF extracts expressing D256A variant of Polβ and supplemented with 0.5 pmol of recombinant Polβ or Polλ. Data points from experiment D256A-Polβ expressing extract are also plotted for reference. The means ± standard errors are plotted (n=3).

Article Snippet: Full-length human Polλ protein and rabbit anti-Polλ polyclonal antibody were kind gifts from Drs. Thomas Kunkel and Samuel H. Wilson respectively.

Techniques: Expressing, Variant Assay, Recombinant

Journal: Journal of internal medicine

Article Title: Elevated expression of CX3C chemokine receptor 1 mediates recruitment of T cells into bone marrow of patients with acquired aplastic anaemia.

doi: 10.1111/joim.12218

Figure Lengend Snippet: Fig. 5 Chemotaxis and adhesion of T cells. Representative images (a) and quantification of chemotaxis of peripheral blood CD4+ (b) and CD8+ (c) T cells from patients with severe aplastic anaemia (SAA: n = 17) and 10 healthy control subjects (n = 10) in response to autologous bone marrow plasma or soluble fractalkine (CX3CL1) in the absence or presence of the CX3C chemokine receptor 1 antagonist F1 or anti-CX3CL1 neutralizing monoclonal antibodies. Results are expressed as chemotactic index (CI). Representative images (d) and quantification of adhesion of peripheral blood CD4+ (e) and CD8+ (f) T cells in response to immobilized CX3CL1 in the absence or presence of F1. Random adhesion was subtracted, and data were expressed as the mean number of cells per field of view. Data are presented as mean SD. Comparisons between different groups: *P < 0.05, **P < 0.01.

Article Snippet: In brief, the lower wells were filled with either 500 lL of autologous bone marrow plasma collected from SAA patients or healthy control subjects, or 500 lL of chemoattractant solution containing assaymedium (RPMI 1640 with 0.5% BSA, 20 mmol L 1 HEPES, pH 7.4) and human CX3CL1 (chemokine domain full length with a poly-His-tail, R&D Systems) at concentrations known to be chemotactic for T cells and monocytes [24].

Techniques: Chemotaxis Assay, Control, Clinical Proteomics, Bioprocessing

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: Tmem184a MO Treatment Results in Tmem184a Protein Decrease. Embryos were injected with SC or Tmem184a ATG MO and harvested for western blotting. Three identically treated embryos constituted a single sample. One of three identical experiments is shown with the graph summarizing the data.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Injection, Western Blot

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: Tmem184a KD Results in Decreased Number of Intact Intersegmental Vessels. (A,A′) SC MO injected embryo (n = 35). (B,B′) Tmem184a SB MO injected embryo (n = 25). (C,C′) Tmem184a ATG MO injected embryo (n = 19). Asterisks indicate ISVs that failed to complete injected embryos. (D,D′) Injection with full-length Tmem184a mRNA alone (n = 21). (E,E′) Co-injection with full-length Tmem184a mRNA rescues vascular defects in Tmem184a SB MO injected embryos (n = 15). (A′–E′) illustrate zoomed trunk regions of the fish. (F) . KD by SB and ATG MOs compared to SC MO embryos and rescue embryos with SB MO and Tmem184a mRNA, illustrating the mean number of intact ISVs ± SEM. p < 0.001. (G) Embryo survival for all treatments shown as compared to SC embryo survival at 24 h ± SEM p < 0.001.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Injection

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: Removing HS binding domains negatively affects Angiogenesis. Embryos were injected with (A,A′) SC MO (n = 20) (C,C′) 25 ng/μL tmem184a ΔCT (n = 60) or (D,D′) 100 ng/µL tmem184a ΔCT (n = 60) and evaluated at 48 hpf as described in Methods. SB results are shown for comparison (B,B′) . (A′′–D′′) illustrate brightfield images from identically treated embryos. Graph (E) illustrates the intact ISVs in SC, tmem184a -ΔCT at either concentration. p < 0.001 (F) The distance between ISVs was measured for each treatment using Image J. P < 0.001 (G) Embryo survival for all treatments illustrated relative to 24 h survival of SC embryos.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Binding Assay, Injection, Comparison, Concentration Assay

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: tmem184a-∆CT mRNA does not rescue knockdown phenotypes. Embryos were injected with (A,A′) SC MO (n = 20) (B,B′) SB MO followed by 25 ng/μL full-length Tmem184a mRNA (n = 60) (C,C′) SB MO (n = 40) (D,D′) SB MO followed by 25 ng/μL tmem184a-ΔCT (n = 60) or (E,E′) SB MO followed by 100 ng/µL tmem184a-ΔCT (n = 60) and evaluated at 48 hpf as described in Methods. (F) The graph illustrates the intact ISVs in SC, rescue (SB MO plus full-length Tmem184a mRNA as in ) embryos, SB MO plus tmem184a-ΔCT at either concentration. p < 0.001. (G) The distance between ISVs was measured for each treatment using Image J. P < 0.001. (H) Embryo survival for all treatments illustrated relative to 24 h survival of SC embryos.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Knockdown, Injection, Concentration Assay

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: Tmem184a and Vegfr2 Function Synergistically to Modulate Angiogenesis. (A,A′) Standard control MO injected embryos (n = 35). (B,B′) Vegfr2 MO injected embryos (n = 20). Asterisks indicate ISVs that have failed to complete growth. (C,C′) Embryos injected with the subthreshold concentration of the Tmem184a ATG MO (0.50 mM) (n = 14). (D,D′) Embryos injected with the subthreshold concentration of the Vegfr2 MO (0.25 mM) (n = 8). (E,E′) 48 hpf embryos co-injected with both subthreshold concentrations of the Tmem184a ATG and Vegfr2 MOs (n = 37). Asterisks indicate ISVs that failed to complete growth. (F) This graph illustrates the mean number of intact ISVs ± SEM in 1 mm Vegfr2 and SC MOs. p < 0.01. (G) The graph is the mean number of intact ISVs ± SEM in subthreshold injected Tmem184a ATG and Vegfr2 MOs as compared to both subthreshold MOs. p < 0.01. (H) Embryo survival for all treatments illustrated relative to 24 h survival of SC embryos.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Control, Injection, Concentration Assay

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: Tmem184a KD Causes an Increase in Cell Proliferation. (A) Example images of PH3 staining in SC, Tmem184a, and Vegfr2 MO knockdown embryos at 48 hpf. White arrows indicate where the PH3 staining co-localizes with the GFP in the vasculature of the ATG MO-treated embryo, highlighted in yellow. Z-stack images were rotated to confirm nuclear PH3 in the EC. Embryos injected with the Tmem184a MO (n = 21) exhibit an increase in the percentage of ISVs that contain an PH3 stained nucleus compared to either SC (n = 21) or Vegfr2 (n = 11) injected embryos. (B) One PH3 nucleus in an ISV is shown in all planes. Cross-hairs intersect at the stained ISV localized nucleus. (C) The graph illustrates the percentage of ISVs per embryo containing an PH3-stained nucleus. p < 0.001.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Staining, Knockdown, Injection

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: KD of Tmem184a results in decreased levels of VE-cadherin. Embryos were injected with control or ATG-MO for Tmem184a, sorted for GFP positive and fixed at 43 hpf. Embryos were stained with the Anti-zebrafish VE-cadherin antibody as described in Methods and imaged on the Zeiss LSM 880 confocal microscope at 20x. (A) Sections in the mid-section of representative embryos are illustrated. Sections illustrated are maximum intensity images as in Methods. Data from 34 Tmem184a KD ISVs and 29 control ISVs from four embryos of each type were analyzed for VE-cadherin intensity across all ISVs in the mid-sections imaged and a graph illustrating relative VE-cadherin is shown in (B) . A student’s T test was used to determine significance, p < 0.001 (C) Western blots of identically treated embryo lysates are shown.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Injection, Control, Staining, Microscopy, Western Blot

Journal: Frontiers in Physiology

Article Title: Involvement of transmembrane protein 184a during angiogenesis in zebrafish embryos

doi: 10.3389/fphys.2022.845407

Figure Lengend Snippet: Model. Based on the literature, we hypothesized HS interaction with Tmem184a is involved in modulation of Vegfr2 signal transduction. VEGF binding to Vegfr2 induces a signaling cascade responsible for the EC survival, permeability, migration, and proliferation necessary for angiogenesis. The present study provides evidence supporting this hypothesis.

Article Snippet: Full length mRNA (i.e., 5′UTR plus coding sequence) encoding 420 amino acids and tmem184a-ΔCT mRNA (i.e., full length minus coding for the final 100 amino acids—referred to as the C-terminal domain) for Tmem184a were synthesized in the pcDNA3.+ plasmid by GenScript.

Techniques: Transduction, Binding Assay, Permeability, Migration

Journal: Gene

Article Title: MUL1 identified as mitochondria-linked biomarker promoting cisplatin resistance in OC cells.

doi: 10.1016/j.gene.2024.148841

Figure Lengend Snippet: Fig. 7. MUL1 enhances platinum resistance in A2780 cell line through apoptosis inhibition. (A) Correlation between MUL1 expression level and platinum resistance. (B) IHC images of MUL1 in normal ovarian tissue. (C) IHC images of MUL1 in OC tissue. Scale: 50 um. (D) Transfection efficiency determined by Western blot. (E) Protein expression of apoptosis markers PARP1, and BCL2 in MUL1 control, knockdown, and overexpression groups. (F) CCK-8 proliferation assay of MUL1 control and knockdown groups at increasing concentrations of cisplatin. **P <0.01. original blots/gels are presented in Supplementary figures 4-10.

Article Snippet: Antibody: PARP1 (proteintech 66520-1-IG, full length weighs 113 kDa), GAPDH (abcam ab8245, weighs 36 kDa), BCL2 (proteintech 12789-1-AP, weighs 26 kDa), MUL1 (proteintech 16133-1-AP, weighs 35 kDa), β-actin (abcam ab8227, weighs 43 kDa).

Techniques: Inhibition, Expressing, Transfection, Western Blot, Control, Knockdown, Over Expression, CCK-8 Assay, Proliferation Assay