entry module ptwist c atg5 d  (Twist Bioscience)

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a . Confocal microscopy images of the trichoblasts and atrichoblasts in the root maturation zone of Arabidopsis wildtype Col-0 expressing ProUBQ10:GFP-ATG8A–I isoforms under nitrogen-starvation conditions. 5 days old Arabidopsis seedlings were incubated in nitrogen-deficient 1/2 MS media containing 2 μM concanamycin A for 2 h before imaging. Representative images of 5 replicates are shown. Area highlighted in the white-boxed region in the GFP-ATG8 + PI panel was further enlarged and presented in the inset panel. Scale bars, 30 μm. Inset scale bars, 10 μm. Green color, GFP-ATG8A–I isoforms. Magenta color, propidium iodide dye. T, trichoblasts. A, atrichoblasts. b . Quantification of the GFP-ATG8 puncta inside the vacuole per normalized area (10,000 μm 2 ) of the trichoblasts and atrichoblasts imaged in a . Bars indicate the mean ± SD of 5 replicates. Two-tailed and paired student t tests were performed to analyze the significance of GFP-ATG8 puncta density differences between the trichoblasts and the atrichoblasts. Exact p values are centered directly above the comparison brackets. c . Confocal microscopy images of trichoblasts and atrichoblasts of Arabidopsis wildtype Col-0 expressing ProATG8E:GFP-ATG8E. 5 days old Arabidopsis seedlings were incubated in 1/2 MS media containing 2 μM conA for 3 h before imaging. Representative images of 5 replicates are shown. Area highlighted in the white-boxed region in the GFP-ATG8E + PI panel was further enlarged and presented in the inset panel. Scale bars, 30 μm. Inset scale bars, 10 μm. Green color, GFP-ATG8E. Magenta color, propidium iodide dye. T, trichoblasts. A, atrichoblasts. d . Quantification of the GFP-ATG8E puncta inside the vacuole per normalized area (10,000 μm 2 ) of the trichoblasts and atrichoblasts imaged in c . Bars indicate the mean ± SD of 5 replicates. Two-tailed and paired student t test was performed to analyze the significance of GFP-ATG8E puncta density difference between the trichoblasts and the atrichoblasts. Exact p values are centered directly above the comparison brackets. e . Confocal microscopy images of epidermal cells in the root maturation zone of Col-0, atg5 , rhd6 rsl1 , cpc try , gl2 and wer myb23 expressing ProUBQ10:GFP-ATG8A under nitrogen-starvation conditions. 5-d-old Arabidopsis seedlings were incubated in nitrogen-deficient 1/2 MS media containing 2 μM concanamycin A for 2 h before imaging. Representative images of 5 replicates are shown. Area highlighted in the white-boxed region in the GFP-ATG8A + PI panel was further enlarged and presented in the inset panel. Scale bars, 30 μm. Inset scale bars, 10 μm. Green color, GFP-ATG8A. Magenta color, propidium iodide dye. A, the atrichoblast positions (adjacent to only one cortex cell). Note that the spindle-shaped ER bodies in wer myb23 (white arrow) were not inside the vacuole and were not counted as GFP-ATG8A puncta for autophagic flux quantification. f . Quantification of the GFP-ATG8A puncta inside the vacuole per normalized area (10,000 μm 2 ) of the cells at the trichoblast positions and the atrichoblast positions imaged in e . Bars indicate the mean ± SD of 5 replicates. Paired repeated measures one-way ANOVA and Fisher’s LSD tests were used to analyze the differences of the number of the GFP-ATG8A puncta between each group. Groups sharing the same letters (a/b/c) are not significantly different, while those with different letters are significantly different. Family-wise significance and confidence level, 0.05 (95% confidence interval).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Confocal Microscopy, Expressing, Incubation, Imaging, Two Tailed Test, Comparison

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a , Trichoblast–atrichoblast distribution patterns in Arabidopsis wild-type Col-0, the autophagy-defective mutant atg5 and the root-hair-development mutant lines rhd6 rsl1 , cpc try , gl2 and wer myb23 . b , c , Confocal microscopy images of epidermal cells in the root maturation zone of Col-0, atg5 , rhd6 rsl1 , cpc try , gl2 and wer myb23 expressing ProUBQ10 :: GFP-ATG8A under control ( b ) or NaCl ( c ) treatment. Five-day-old Arabidopsis seedlings were incubated in either control 1/2 MS media containing 2 μM conA for 2 h ( b ) or 1/2 MS media containing 50 mM NaCl + 1 μM conA for 40–60 min ( c ) before imaging. Representative images of five replicates are shown. The areas highlighted by white boxes in the GFP–ATG8A + PI panels are further enlarged and presented in the inset panels. Scale bars, 30 μm. Inset scale bars, 10 μm. Green indicates GFP–ATG8A; magenta indicates PI dye. T, trichoblast position (adjacent to two cortex cells); A, atrichoblast position (adjacent to only one cortex cell). Note that the spindle-shaped ER bodies in wer myb23 (white arrows in c ) were not inside the vacuole and were not counted as GFP–ATG8A puncta for autophagic flux quantification. d , e , Quantification of GFP–ATG8A puncta inside the vacuole per normalized area (10,000 μm 2 ) of the cells at the trichoblast positions and the atrichoblast positions imaged in b ( d ) or c ( e ). The bars indicate the mean ± s.d. of five replicates. Paired repeated-measures one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) tests were used to analyse the differences in the numbers of GFP–ATG8A puncta between each group. Groups sharing the same letters (a/b/c/d/e) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Mutagenesis, Confocal Microscopy, Expressing, Control, Incubation, Imaging

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a , Design of the trichoblast-specific ATG5 CRISPR mutagenesis plasmid expressed in wild-type Col-0 expressing ProUBQ10 :: mCherry-ATG8E ( mCherry-ATG8E in Col-0). b , Design of the trichoblast-specific ATG5 complementation plasmid transformed to complement the autophagy-defective mutant atg5 expressing ProUBQ10 :: mCherry-ATG8E ( mCherry-ATG8E in atg5 ). c , d , Confocal microscopy images of trichoblasts and atrichoblasts of Arabidopsis lines mCherry-ATG8E in Col-0, TSKO-R ( ATG5 mutagenized using ProRHD6 -driven Cas9), R-ATG5 (the atg5 mutant complemented with ProRHD6 -driven ATG5 ), E-ATG5 (the atg5 mutant complemented with ProEXP7 -driven ATG5 ) and mCherry-ATG8E in atg5 under control ( c ) or NaCl ( d ) treatment. Five-day-old Arabidopsis seedlings were incubated in either control 1/2 MS media containing 2 μM conA for 2 h ( c ) or 1/2 MS media containing 50 mM NaCl + 1 μM conA for 1 h ( d ) before imaging. Representative images of five replicates are shown. The areas highlighted by white boxes in the merge panels are further enlarged and presented in the inset panels. Scale bars, 30 μm. Inset scale bars, 10 μm. T, trichoblast; A, atrichoblast. e , f , Quantification of mCherry–ATG8E puncta inside the vacuole per normalized area (10,000 μm 2 ) of the trichoblasts and atrichoblasts imaged in c ( e ) or d ( f ). The bars indicate the mean ± s.d. of five replicates. Paired repeated-measures one-way ANOVA and Fisher’s LSD tests were used to analyse the differences in the numbers of mCherry–ATG8E puncta between each group. Groups sharing the same letters (a/b/c) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: CRISPR, Mutagenesis, Plasmid Preparation, Expressing, Transformation Assay, Confocal Microscopy, Control, Incubation, Imaging

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a-d . RHD6 promoter is more efficient in disrupting autophagic flux in trichoblast cells compared to EXP7 promoter. a-b . Confocal microscopy images of trichoblasts and atrichoblasts in the root maturation zone of Arabidopsis lines mCherry-ATG8E in Col-0, TSKO-R ( ATG5 mutagenized using ProRHD6 -driven Cas9), TSKO-E ( ATG5 mutagenized using ProEXP7 -driven Cas9) and mCherry-ATG8E in atg5 under control ( a ) or ( b ) treatment. 5 days old Arabidopsis seedlings were incubated in either 1/2 MS media containing 2 μM concanamycin A for 2 h ( a ) or 1/2 MS media containing 50 mM NaCl + 1 μM concanamycin A for 1 h ( b ) before imaging. Representative images of 5 replicates are shown. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 30 μm. Inset scale bars, 10 μm. T, trichoblasts. A, atrichoblasts. c-d . Quantification of the mCherry-ATG8E puncta inside the vacuole per normalized area (10,000 μm 2 ) of the cells of the trichoblasts and atrichoblasts imaged in a ( c ) or b ( d ). Bars indicate the mean ± SD of 5 replicates. Paired repeated measures one-way ANOVA and Fisher’s LSD tests were used to analyze the differences of the number of the mCherry-ATG8E puncta between each group. Groups sharing the same letters (a/b/c) are not significantly different, while those with different letters are significantly different. Family-wise significance and confidence level, 0.05 (95% confidence interval). e. Normal autophagosome structures can be observed in cells that do not express Cas9 in TSKO-R lines . Confocal microscopy images of cells of cotyledon epidermis and stele regions of TSKO-R. 5 days old Arabidopsis seedlings were incubated in control 1/2 MS media for 45 min before imaging. Representative images of 3 replicates are shown. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 20 μm. Inset scale bars, 5 μm.

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Confocal Microscopy, Control, Incubation, Imaging

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a . Confocal microscopy images showing sodium ion concentrations in the vacuoles of trichoblasts and atrichoblasts in the transition zone and the elongation zone of Arabidopsis wildtype Col-0 and the autophagy-defective mutants atg5 , atg8 and atg16 indicated by CoroNa Green AM staining. 5 days old Arabidopsis seedlings were incubated in control 1/2 MS media for 30 min and were subsequently incubated in control 1/2 MS media containing 2 μM CoroNa Green AM for 30 min before imaging. Representative images of 5 replicates are shown. Scale bars, 20 μm. Green color, CoroNa Green AM sodium indicator. Magenta color, propidium iodide dye. T, trichoblasts. A, atrichoblasts. b-c . Quantitative analysis of the vacuolar CoroNa Green AM fluorescence intensity ratio between trichoblasts and atrichoblasts at the transition zone ( b ) or elongation zone ( c ) of the Arabidopsis seedlings imaged in a . Bars indicate the mean ± SD of 5 replicates. Paired repeated measures one-way ANOVA and Fisher’s LSD tests were performed to analyze the significance of the CoroNa Green AM fluorescence intensity ratio differences between each group. Groups sharing the same letter ‘a’ are not significantly different. Family-wise significance and confidence level, 0.05 (95% confidence interval). d . Confocal microscopy images showing sodium ion concentrations in the vacuoles of trichoblasts and atrichoblasts in the transition zone and the elongation zone of Arabidopsis lines mCherry-ATG8E in Col-0, TSKO-R, R-ATG5, E-ATG5 and mCherry-ATG8E in atg5 indicated by CoroNa Green AM staining. 5 days old Arabidopsis seedlings were incubated in control 1/2 MS media for 30 min and were subsequently incubated in control 1/2 MS media containing 2 μM CoroNa Green AM for 30 min before imaging. Representative images of 5 replicates are shown. Scale bars, 20 μm. Green color, CoroNa Green AM sodium indicator (and the nuclear signals of Cas9-GFP in TSKO-R). Magenta color, mCherry-ATG8E. T, trichoblasts. A, atrichoblasts. e-f . Quantitative analysis of the vacuolar CoroNa Green AM fluorescence intensity ratio between trichoblasts and atrichoblasts in the transition zone ( e ) or elongation zone ( f ) of the Arabidopsis seedlings imaged in d . Bars indicate the mean ± SD of 5 replicates. Paired repeated measures one-way ANOVA and Fisher’s LSD tests were performed to analyze the significance of the CoroNa Green AM fluorescence intensity ratio differences between each group. Groups sharing the same letter ‘a’ are not significantly different. Family-wise significance and confidence level, 0.05 (95% confidence interval).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Confocal Microscopy, Staining, Incubation, Control, Imaging, Fluorescence

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a , Confocal microscopy images showing sodium ion accumulation in the vacuoles of trichoblasts and atrichoblasts in the root maturation zone of Arabidopsis wild-type Col-0 and the autophagy-defective mutants atg5 , atg8 and atg16 . Five-day-old Arabidopsis seedlings were incubated in control 1/2 MS media for 30 min and were subsequently incubated in control 1/2 MS media containing 2 μM CoroNa Green AM for 30 min before imaging. Representative images of five replicates are shown. The areas highlighted by white boxes in the CoroNa Green AM + PI panels are further enlarged and presented in the inset panels. Scale bars, 30 μm. Inset scale bars, 10 μm. Green indicates CoroNa Green AM sodium indicator; magenta indicates PI dye. T, trichoblast; A, atrichoblast. b , Quantitative analysis of the vacuolar CoroNa Green AM fluorescence intensity ratio between trichoblasts and atrichoblasts of the Arabidopsis seedlings imaged in a . The bars indicate the mean ± s.d. of five replicates. Paired repeated-measures one-way ANOVA and Fisher’s LSD tests were performed to analyse the significance of the CoroNa Green AM fluorescence intensity ratio differences between each group. Groups sharing the same letters (a/b) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval). c , Phenotypic characterization of the seedlings of Arabidopsis wild-type Col-0 and the autophagy-defective mutants atg5 , atg8 and atg16 under NaCl stress treatment. Representative images of three replicates are shown. d , Quantitative analysis of the proportion of non-etiolated leaves of the Arabidopsis seedlings imaged in c . The bars indicate the mean ± s.d. of three replicates. Paired repeated-measures one-way ANOVA and Fisher’s LSD tests were performed to analyse the significance of the proportion differences between each group. Groups sharing the same letters (a/b) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval). e , Confocal microscopy images showing the sodium ion concentrations in the vacuoles of trichoblasts and atrichoblasts in the root maturation zone of Arabidopsis lines mCherry-ATG8E in Col-0, TSKO-R, R-ATG5, E-ATG5 and mCherry-ATG8E in atg5 . Five-day-old Arabidopsis seedlings were incubated in control 1/2 MS media for 30 min and were subsequently incubated in control 1/2 MS media containing 2 μM CoroNa Green AM for 30 min before imaging. Representative images of five replicates are shown. The areas highlighted by white boxes in the CoroNa Green AM + mCherry–ATG8E panels are further enlarged and presented in the inset panels. Scale bars, 30 μm. Inset scale bars, 10 μm. Green indicates CoroNa Green AM sodium indicator (and the nuclear signals of Cas9–GFP in TSKO-R); magenta indicates mCherry–ATG8E. T, trichoblast; A, atrichoblast. f , Quantitative analysis of the vacuolar CoroNa Green AM fluorescence intensity ratio between trichoblasts and atrichoblasts of the Arabidopsis seedlings imaged in e . The bars indicate the mean ± s.d. of five replicates. Paired repeated-measures one-way ANOVA and Fisher’s LSD tests were performed to analyse the significance of the CoroNa Green AM fluorescence intensity ratio differences between each group. Groups sharing the same letters (a/b) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval). g , Phenotypic characterization of the seedlings of Arabidopsis lines mCherry-ATG8E in Col-0, TSKO-R, R-ATG5, E-ATG5 and mCherry-ATG8E in atg5 under NaCl stress treatment. Representative images of ten replicates are shown. h , Quantitative analysis of the proportion of non-etiolated leaves of the Arabidopsis seedlings imaged in g . The bars indicate the mean ± s.d. of ten replicates. Paired repeated-measures one-way ANOVA and Fisher’s LSD tests were performed to analyse the significance of the proportion differences between each group. Groups sharing the same letters (a/b/c/d) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Confocal Microscopy, Incubation, Control, Imaging, Fluorescence

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a-b . Quantitative analysis of primary root length of Arabidopsis seedlings imaged in Fig. ( a , n = 24) or Fig. ( b , n = 70). Repeated measures one-way ANOVA and Tukey tests were performed to analyze the significance of the primary root elongation difference between each group. Groups sharing the same letter ‘a’ are not significantly different. Family-wise significance and confidence level, 0.05 (95% confidence interval). Black lines indicate the median. White lines, 25% and 75% quartiles. c-d . Quantitative analysis of the number of newly emerged lateral roots of Arabidopsis seedlings imaged in Fig. ( a , n = 24) or Fig. ( b , n = 70). Unpaired Brown-Forsythe and Welch ANOVA tests and two-sided unpaired t tests with Welch’s correction (for multiple comparison) were performed to analyze the significance of the number of newly emerged lateral roots between each group, n = 24. Groups sharing the same letters (a/b/c/d) are not significantly different, while those with different letters are significantly different. Family-wise significance and confidence level, 0.05 (95% confidence interval). Black lines indicate the median. White lines, 25% and 75% quartiles. Note that the median and 25% quartile lines of atg5 , atg8 , and atg16 were all at the base line (y = 0), and the median line of mCherry-ATG8E in atg5 , the 25% quartile lines of R-ATG5, E-ATG5 and mCherry-ATG8E in atg5 were all at the base line (y = 0).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Comparison

Journal: Nature Plants

Article Title: Cell-type-specific autophagy in root-hair-forming cells is essential for salt stress tolerance in Arabidopsis thaliana

doi: 10.1038/s41477-026-02285-w

Figure Lengend Snippet: a , Confocal microscopy images showing the relative ROS levels in the trichoblasts and atrichoblasts of Arabidopsis wild-type Col-0 and the autophagy-defective mutants atg5 , atg8 and atg16 . Five-day-old Arabidopsis seedlings were incubated in either 1/2 MS (control) or 1/2 MS + 50 mM NaCl (NaCl) liquid media for 45 min and were subsequently stained with 1 μM CM-H 2 DCFDA for 1 min before imaging. Representative images of five replicates are shown. The areas highlighted by white boxes in the CM-H 2 DCFDA panels are further enlarged and presented in the inset panels. Scale bars, 30 μm. Inset scale bars, 30 μm. T, trichoblast; A, atrichoblast. c , Confocal microscopy images showing the relative ROS levels in the trichoblasts and atrichoblasts of Arabidopsis lines mCherry-ATG8E in Col-0, TSKO-R, R-ATG5, E-ATG5 and mCherry-ATG8E in atg5 . Five-day-old Arabidopsis seedlings were incubated in either 1/2 MS (control) or 1/2 MS + 50 mM NaCl (NaCl) liquid media for 45 min and were subsequently stained with 1 μM CM-H 2 DCFDA for 1 min before imaging. Representative images of five replicates are shown. The areas highlighted by white boxes in the CM-H 2 DCFDA panels are further enlarged and presented in the inset panels. Scale bars, 30 μm. Inset scale bars, 30 μm. b , d , Quantitative analysis of the relative ROS ratio between trichoblasts and atrichoblasts of the Arabidopsis seedlings imaged in a ( b ) or c ( d ). The bars indicate the mean ± s.d. of five replicates. Paired repeated-measures two-way ANOVA and Fisher’s LSD tests were performed to analyse the significance of the CM-H 2 DCFDA fluorescence intensity ratio differences between each group. Groups sharing the same letter (a/b/c) are not significantly different, while those with different letters are significantly different. The family-wise significance and confidence level was 0.05 (95% confidence interval).

Article Snippet: The coding sequence of Arabidopsis ATG5 (Supplementary Table ) was cloned into pTwist Amp High Copy to form the entry module pTwist-C- ATG5 -D (Twist Bioscience).

Techniques: Confocal Microscopy, Incubation, Control, Staining, Imaging, Fluorescence

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