ultimate tol2 integrable construct  (Addgene inc)

Journal: Journal of Translational Medicine

Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

doi: 10.1186/s12967-026-07680-7

Figure Lengend Snippet: RT induces LUAD cell radio-resistance by evoking MTHFD2 overexpression. A Baseline MTHFD2 expression: left: immunoblots of MTHFD2 protein levels across LUAD cell lines. Right: densitometric quantification normalized to β-tubulin. B Left: Representative colony formation images post-X-ray irradiation (0–4 Gy). Right: cell survival curves. ( C - D ) Radiation-induced MTHFD2 regulation: ( C ) time-course immunoblot (0–48 h post-6 Gy) demonstrating temporal coordination of MTHFD2 and γ-H2AX expression. ( D ) dose-dependent upregulation (0–10 Gy) of MTHFD2 and DNA damage markers. E Nuclear/cytoplasmic fractionation showing radiation-triggered (6 Gy) MTHFD2 nuclear accumulation over time (lamin a/c nuclear marker, GAPDH cytoplasmic control). F Schematic of iterative fractionated irradiation (2 Gy × 30 fractions) for generating radioresistant clones. G Left: immunoblots of MTHFD2 and γ-H2AX in parental vs. radioresistant lines. Middle and right: quantitative comparison showing MTHFD2 elevation and γ-H2AX reduction in resistant clones. H DNA repair capacity assessment: left: neutral comet assay images post-6 Gy irradiation. Right: olive tail moment quantification demonstrating impaired DNA repair in radioresistant cells (scale bar: 50 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p< 0.001，*** p< 0.001, **** p < 0.0001)

Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Over Expression, Expressing, Western Blot, Irradiation, Fractionation, Marker, Control, Clone Assay, Comparison, Neutral Comet Assay, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

doi: 10.1186/s12967-026-07680-7

Figure Lengend Snippet: MTHFD2 interacts with XRCC6 and controls XRCC6 expression. A Venn diagram illustrating the overlap between MTHFD2-associated proteins identified by LC-MS/MS analysis ( n = 1,247) and curated DNA repair factors (HR/NHEJ pathways). Six candidate interactors are highlighted. B Co-IP validation of MTHFD2-binding partners in H1299 cells using anti-flag antibody. C Co-immunoprecipitation assay detection of MTHFD2, SMC6 and XRCC6 with endogenous MTHFD2 in whole-cell lysates. D Co-immunoprecipitation assay detection of MTHFD2, SMC6 and XRCC6 with endogenous SMC6 and XRCC6 in whole-cell lysates. E Correlation between MTHFD2 and XRCC6 expression levels in radiotherapy-sensitive (case 1) versus radiotherapy-resistant (case 2) LUAD cohorts. Scale bars: 100 μm/25 μm(inset). F XRCC6 protein levels were positively regulated in cells with MTHFD2 deletion or overexpression. G XRCC6 perturbation (knockdown or overexpression) failed to alter MTHFD2 protein abundance. H Co-IP analysis of FLAG-MTHFD2/XRCC6 interaction dynamics in MTHFD2-manipulated LUAD cells (β-tubulin as loading control). I Co-IP confirming interaction specificity: XRCC6 antibody pulls down exogenous MTHFD2 in overexpression models. J in vivo functional correlation: MTHFD2 depletion (shRNA) impairs XRCC6 expression in xenograft tumours post-ionizing radiation treatment, as shown by dual immunofluorescence test. Scale bars: 100 μm/25 μm(inset). Statistical significance determined by two-tailed Student’s t-test ( n = 3, ** p < 0.01)

Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Biomarker Discovery, Binding Assay, Over Expression, Knockdown, Quantitative Proteomics, Control, In Vivo, Functional Assay, shRNA, Immunofluorescence, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

doi: 10.1186/s12967-026-07680-7

Figure Lengend Snippet: XRCC6 overexpression partially rescues MTHFD2 knockdown-induced functional impairment. A Immunoblot analysis of MTHFD2 and XRCC6 expression in LUAD cells transfected with specific shRNA or overexpression constructs. B Cell viability rescue: Cell Counting Kit-8 (CCK-8) assays demonstrated that XRCC6 overexpression restored cellular viability in MTHFD2-silenced cells. C Clonogenic recovery: quantitative colony formation assays confirmed partial reversal of proliferation defects by XRCC6 reconstitution. D DNA damage mitigation: γ-H2AX immunoblotting post-6 Gy irradiation showed XRCC6 overexpression reduced DNA damage signaling. E Comet assay validation: neutral comet quantification revealed XRCC6 reconstitution shortened olive tail moments in MTHFD2-deficient cells (scale bar: 50 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p < 0.01)

Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Over Expression, Knockdown, Functional Assay, Western Blot, Expressing, Transfection, shRNA, Construct, Cell Counting, CCK-8 Assay, Irradiation, Single Cell Gel Electrophoresis, Biomarker Discovery, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

doi: 10.1186/s12967-026-07680-7

Figure Lengend Snippet: MTHFD2 recruits the LIG4/XRCC4/XLF complex to activate DNA damage repair pathways. A Immunoblot analysis of key NHEJ components (MTHFD2, XRCC6, XLF, XRCC4, LIG4) in LUAD cells transfected with specific shRNA. B Immunofluorescence revealed that MTHFD2 depletion markedly attenuated nuclear foci formation of LIG4/XRCC4/XLF complexes (Scale bar: 20 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p < 0.01)

Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Western Blot, Transfection, shRNA, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-TET1-P2A-GFP (LSL-dC-T) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of GFP, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-TET1-GFP mice and LSL-dCas9-TET1-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-TET1-GFP and LSL-dCas9-TET1-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, eGFP, dCas9 colocalization in mice injected contralaterally with either mCherry or mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-TET1 induction efficiency in mCherry-Cre and mCherry-labeled cells. ( n = 3 mice per group, two-sided t test, P = 1.2 × 10 −7 ). F Quantification of the percentage of NeuN+ cells in mCherry− and mCherry+ populations. ( n = 8 mice per group, two-sided t test, P = 2.81 × 10 −8 ).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling