Journal: bioRxiv

Article Title: Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

doi: 10.64898/2026.01.15.699632

Figure Lengend Snippet: MMEJ induced by Cas9 and Cas9n exhibits distinct genetic dependence. ( A-C ) MMEJ was assayed in U2OS (EGFP-MMEJ) cells expressing shRNAs for Polθ ( A ), LIG3 ( A ), RPA2 ( B ), MRE11 ( C ) or CtIP ( C ) after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( D ) MMEJ was assayed in WT or POLQ -KO mES (EGFP-MMEJ) cells four days after transfection of plasmids encoding g2/Cas9 WT or g2/Cas9 D10A . ( E ) mES (EGFP-MMEJ) cells with POLQ WT allele or knock-in (KI) alleles containing mutations of K120G or D2494P/E2495R were assayed for MMEJ after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( F ) In vitro biochemical assay showing Polθ-HelD activity in unwinding dsDNA to facilitate strand exchange with ssDNA carrying 15 bp homology. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrate and incubated with Polθ-HelD, ATP and/or RPA, and the reaction products were resolved on a non-denaturing gel. ( G ) In vitro biochemical assay showing strand displacement DNA synthesis by Polθ-HelD and Polθ-PolD. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrates and incubated with Polθ-HelD, ATP and/or RPA, followed by DNA extension with Polθ-PolD with 0.1 mM dNTPs, and the reaction products were resolved on a denaturing gel.

Article Snippet: Antibodies used in immunoblotting are: MRE11 (Cell Signaling Technology, 4895), CtIP (Proteintech, 12624-1-AP), RPA1 (Sigma-Aldrich, NA13), RPA2 (Bethyl, A300-244A), PCNA (Cell Signaling Technology, 13110), MCM2 (Proteintech, 10513-1-AP), DNA2 (Proteintech, 18727-1-AP), KU70 (Santa Cruz Biotechnology, sc-17789), HA (Santa Cruz Biotechnology, sc-7392), Flag (Sigma-Aldrich, F1804), BLM (Bethyl, A300-110A), EXO1 (Bethyl, A302-640A), ATR (Santa Cruz Biotechnology, sc-515173), BRCA1 (Santa Cruz Biotechnology, sc-6954), RAD51 (Santa Cruz Biotechnology, sc-398587), LIG3 (Proteintech, 26583-1-AP), γH2AX (Upstate, 07-164), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, 115-035-146), and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, 111-035-144).

Techniques: Expressing, Transfection, Knock-In, In Vitro, Activity Assay, Labeling, Incubation, DNA Synthesis

Journal: Oxidative medicine and cellular longevity

Article Title: Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels.

doi: 10.1155/2018/6956414

Figure Lengend Snippet: Figure 9: Western blot analysis of mitochondrial extracts from M-LPH-WT and -KO HepG2 cells. (a) The intramitochondrial levels of proteins essential for mtDNA stability and maintenance were examined using SDS-PAGE gels. Analysis was performed using the membrane extract (ME) used in Figure 2(b). VDAC was used as a loading control for mitochondrial protein. L and S in the figure correspond to the two mitochondrial isoforms of LIG3 generated by the alternative splicing. Three preparations of the membrane extract from WT and KO cells, respectively, were used for analysis. The bars represent the mean ± SD of the results from three independent experiments. Differences at p < 0 05 were considered to be statistically significant (∗p = 0 016, ∗∗p = 0 00018, ∗∗∗p = 0 0075, ∗∗∗∗p = 0 0091, and ∗∗∗∗∗p = 0 03). (b) Western blot analysis using Zn2+-Phos-tag gels was performed to investigate the state of phosphorylation of TFAM. In Zn2+-Phos-tag gel electrophoreses, phosphorylated proteins exhibit lower electrophoretic mobility than do corresponding dephosphorylated proteins [27]. In the presence of 50 μM Phos-tag, the Rf values for TFAM band in M-LPH-KO cells were smaller than those in M-LPH-WT cells. Numbers in graphs represent the intensity of each band. (c) Mitochondrial extracts from M-LPH-WT and -KO cells were incubated with or without 40 U/μl of λ protein phosphatase and analyzed by Zn2+-Phos-tag SDS-PAGE followed by immunoblotting with the anti-TFAM antibody.

Article Snippet: SDSPAGE electrophoresis and Western blot analysis were performed as described previously [26] using anti-M-LPH (HPA041180) and anti-LIG3 (HPA006723) antibodies purchased from Atlas Antibodies (Stockholm, Sweden), anti-voltage-dependent anion channel (VDAC, 55249-1- AP), anti-histone deacetylase 1 (HDAC1, 10197-1-AP), anti-histone cluster 1 H1 family member C (HIST1H1C, 15446-1-AP), and anti-Lon protease-like protein (LONP1, 15440-1-AP) antibodies from Proteintech (Chicago, IL), anti-TFAM (#8076) and anti-DNA polymerase γ, catalytic subunit (POLG, #13609) antibodies from Cell Signaling Technology (Danvers, MA), anti-OGG1 antibody (NB100106) from Novus Biologicals (Littleton, CO), anti-heat shock protein 90 alpha family class A member 1 (HSP90AA1, GTX109753) and anti-gamma H2A histone family member X (γ-H2AX, GTX11174) antibodies from GeneTex (Irvine, CA), anti-Twinkle helicase (TWNK, ab83329) antibody from Abcam (Cambridge, UK), and anti-β-actin antibody (A2066) from Sigma (St. Louis, MO).

Techniques: Western Blot, SDS Page, Membrane, Control, Generated, Alternative Splicing, Phospho-proteomics, Incubation

Journal: Redox Biology

Article Title: CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla

doi: 10.1016/j.redox.2023.102782

Figure Lengend Snippet: Lig3 was a downstream mRNA target of miR-335. ( A ) Schematic graph of the potential binding sites for miR-335 within Lig3 3′ UTR. ( B ) The luciferase activities of Lig3 luciferase reporters (WT or MUT) were tested in B104 cells co-expressing miR-335 agomir or agomir NC. ( C and D ) RT-qPCR and Western blot analyses showed that the expression of Lig3 decreased or increased in B104 cells when miR-335 was overexpressed or downregulated, respectively. ( E ) Following miR-335 knockdown in RVLM of SIH rats, Lig3 expression was measured using RT-qPCR and Western blot assays. Data were expressed as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t -test ( B–D ) and one-way ANOVA, followed by post-hoc Bonferroni test ( E ). n = 3 – 6 of independent cell culture preparations ( B–D ). n = 3 – 6 rats per group ( E ). ## p < 0.01, ### p < 0.001, and ns means nonsignificant versus agomir NC group. Δ p < 0.05, and ΔΔ p < 0.01 versus antagomir NC group. ** p < 0.01, and *** p < 0.001 versus SIH group.

Article Snippet: Afterward, the membranes were blotted with rabbit polyclonal Lig3 (1:1000, ABclonal, China) and mouse monoclonal horseradish peroxidase (HRP)-conjugated GAPDH (1:5000, Proteintech, USA) primary antibodies at 4 °C overnight.

Techniques: Binding Assay, Luciferase, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Two Tailed Test, Cell Culture

Journal: Redox Biology

Article Title: CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla

doi: 10.1016/j.redox.2023.102782

Figure Lengend Snippet: MiR-335 caused oxidative stress in RVLM of SIH rats via targeting Lig3. ( A–C ) Effects of miR-335 downregulation on the level of 8-OH-dG and activities of SOD and CAT in RVLM of SIH rats were reversed by Lig3 depletion. ( D ) Lig3 knockdown weakened the suppressive influence of miR-335 silencing on ROS production in RVLM of SIH rats. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test ( A–D ). n = 6 rats per group ( A–D ). * p < 0.05, ** p < 0.01, *** p < 0.001, and ns means nonsignificant versus SIH group.

Article Snippet: Afterward, the membranes were blotted with rabbit polyclonal Lig3 (1:1000, ABclonal, China) and mouse monoclonal horseradish peroxidase (HRP)-conjugated GAPDH (1:5000, Proteintech, USA) primary antibodies at 4 °C overnight.

Techniques: Knockdown

Journal: Redox Biology

Article Title: CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla

doi: 10.1016/j.redox.2023.102782

Figure Lengend Snippet: CircRNA Galntl6 regulated Lig3 expression via targeting miR-335. ( A and B ) Effects of circRNA Galntl6 overexpression and miR-335 agomir on the expression of Lig3 were revealed using RT-qPCR and Western blot analyses in B104 cells. ( C and D ) RT-qPCR and Western blot assays were used to illustrate the effects of circRNA Galntl6 knockdown and miR-335 antagomir on Lig3 expression in B104 cells. ( E and F ) After microinjection of pLV-circRNA Galntl6 plasmid and miR-335 agomir into RVLM of SIH rats, the expression of Lig3 was measured by RT-qPCR and Western blot analyses. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test ( A–F ). n = 6 of independent cell culture preparations ( A and C ). n = 3 of independent cell culture preparations ( B and D ). n = 6 rats per group ( E ). n = 3 rats per group ( F ). # p < 0.05, ## p < 0.001, and ns means nonsignificant versus pLV-NC group. Δ p < 0.05, ΔΔΔ p < 0.001, and ns means nonsignificant versus si-NC group. * p < 0.05, *** p < 0.001, and ns means nonsignificant versus SIH + pLV-NC group.

Article Snippet: Afterward, the membranes were blotted with rabbit polyclonal Lig3 (1:1000, ABclonal, China) and mouse monoclonal horseradish peroxidase (HRP)-conjugated GAPDH (1:5000, Proteintech, USA) primary antibodies at 4 °C overnight.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Knockdown, Microinjection, Plasmid Preparation, Cell Culture