Journal: Journal of Experimental Botany

Article Title: EARLY BUD BREAK 1 triggers bud break in peach trees by regulating hormone metabolism, the cell cycle, and cell wall modifications

doi: 10.1093/jxb/eraa119

Figure Lengend Snippet: Transient expression of PpEBB1 in peach buds. (A) Unrooted neighbor-joining tree of orthologous genes of PpEBB1 from different species. (B) Transient conversion of PpEBB1 in peach buds with an EBB1 overexpression level three times greater than that in the control. The values represent the means ±SD of three replicates, and the different letters above the bars represent significant differences; P <0.05. (C, D) Comparison of peach buds injected with a PpEBB1-IL60 vector and an empty IL60 vector (as a control). Ninety buds were divided into three groups as three replicates for transformation in each treatment. The proportion of bud break was calculated according to the sum of the buds in the three groups, and the buds that ultimately burst were included in the total buds.

Article Snippet: The full-length cDNA of these genes was inserted into a pGADT7 vector as prey and together with BD-EBB1 1−120aa was used for Y2H assays.

Techniques: Expressing, Over Expression, Injection, Plasmid Preparation, Transformation Assay

Journal: Autophagy

Article Title: CGAS is a micronucleophagy receptor for the clearance of micronuclei

doi: 10.1080/15548627.2021.1899440

Figure Lengend Snippet: CGAS interacted with LC3B. (A) Prediction of LIRs in CGAS by iLIR and protein sequence alignments of conserved LIR motifs. (B) Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293 cells that had been transfected with Flag-LC3B and GFP-CGAS. (C) Results of in vitro precipitation assay with anti-His antibody and protein agarose of His-tagged LC3B and SUMO-tagged CGAS. (D) Binding curves of surface-immobilized SUMO-tagged CGAS with His-tagged LC3B at concentrations of 800 nM, 400 nM, 300 nM, 200 nM and 100 nM. Vertical lines mark the starts of association and dissociation phases of the binding events. The dashed lines are global fits to a Langmuir reaction model; global fitting parameters are listed in the table below the plot. (E) Immunoblot of cell lysates and anti-LC3B immunoprecipitates from U2OS cells in the absence or presence of NDZ (300 nM) treatment for 24 h followed by 24-h recovery, and cells were left untreated or treated with CQ (40 μM) treatment 8 h prior to harvesting. (F and G) PLA showing the interaction of CGAS with LC3B in U2OS cells left untreated or treated with NDZ (300 nM) for 24 h followed by 48-h recovery, and cells were cultured in the absence or presence of CQ (40 μM) for 12 h prior to harvesting (F). The quantification data were shown in (G). Data are representative of n = 3 independent experiments (B-F). Data are expressed as mean ± (SEM) of 3 independent experiments ( G ). Scale bar: 50 μm

Article Snippet: The following primary antibodies were utilized: anti-CGAS rabbit monoclonal antibody (D1D3G; Cell Signaling Technology, 15102), anti-LC3B rabbit polyclonal antibody (MAP1LC3B:1–120aa.

Techniques: Sequencing, Western Blot, Transfection, In Vitro, Binding Assay, Cell Culture

Journal: Autophagy

Article Title: CGAS is a micronucleophagy receptor for the clearance of micronuclei

doi: 10.1080/15548627.2021.1899440

Figure Lengend Snippet: LIR domain of CGAS was required for CGAS-LC3 interaction. (A) Immunoblot of cell lysates and anti-Flag immunoprecipitates from HEK293 cells that had been stably transfected with Flag-LC3B and GFP-CGAS, GFP-CGASΔ355-360 or GFP-CGAS F357A,V360A . (B) Results of in vitro precipitation assay with anti-His antibody and protein agarose of His-tagged LC3B and SUMO-tagged CGAS or SUMO-tagged CGAS F357A,V360A . (C) Immunoblot of cell lysates and anti-LC3B immunoprecipitates from U2OS cells that had been stably transfected with GFP, GFP-CGAS, GFP-CGASΔ355-360 or GFP-CGAS F357A,V360A in the absence or presence of NDZ (300 nM) treatment for 24 h followed by 48-h recovery. Data are representative of n = 3 independent experiments

Article Snippet: The following primary antibodies were utilized: anti-CGAS rabbit monoclonal antibody (D1D3G; Cell Signaling Technology, 15102), anti-LC3B rabbit polyclonal antibody (MAP1LC3B:1–120aa.

Techniques: Western Blot, Stable Transfection, Transfection, In Vitro

Journal: Autophagy

Article Title: CGAS is a micronucleophagy receptor for the clearance of micronuclei

doi: 10.1080/15548627.2021.1899440

Figure Lengend Snippet: CGAS regulated the autophagy of micronuclei. (A) Immunofluorescence assay showing the colocalization of CGAS with LC3B at micronuclei in U2OS cells treated with NDZ (300 nM) for 24 h followed by 48-h recovery, and cells were left treated with CQ (40 μM) for 12 h prior to harvesting. DAPI, nuclei. (B) Immunogold electron microscopy showing the colocalization of CGAS with MN in autophagosome. The red arrow indicated CGAS. (C and D) PLA assay showing the colocalization of CGAS with LC3B at micronuclei in U2OS cells treated with NDZ (300 nM) for 24 h followed by 48-h recovery, and cells were cultured in the absence or presence of CQ (40 μM) for 12 h prior to harvesting (C). The quantification data was shown in (D). (E and F) Immunofluorescent assay showing LC3B and micronuclei of WT and CGAS −/- U2OS cells left untreated or treated with NDZ (300 nM) for 24 h followed by 48-h recovery (E). The quantification data was shown in (F). (G) Immunoblot of cell lysates and lysosomes purified from U2OS cells left untreated or treated with NDZ (300 nM) for 24 h followed by 48-h recovery. (H) Immunofluorescence assay showing the colocalization of CGAS with LC3B and LAMP2 at micronuclei in U2OS cells that had been stably transfected with GFP-CGAS left untreated or treated with NDZ (300 nM) for 24 h followed by 48-h recovery. DAPI, nuclei. (I and J) Immunofluorescent assay showing the staining of LC3B, LAMP2 and micronuclei of WT and CGAS −/- U2OS cells left untreated or treated with NDZ (300 nM) for 24 h followed by 48-h recovery (I). The quantification data is shown in (J). Data are expressed as mean ± (SEM) of 3 independent experiments ( D, F, J ). Data are representative of n = 3 independent experiments ( A-C, E, G-I ). Nuclei were stained with DAPI (blue). One-way ANOVA with Bonferroni post hoc test were used for statistical analysis. Scale bar: 2.5 μm (A and H), 10 μm (C and I), 200 nm ( B )

Article Snippet: The following primary antibodies were utilized: anti-CGAS rabbit monoclonal antibody (D1D3G; Cell Signaling Technology, 15102), anti-LC3B rabbit polyclonal antibody (MAP1LC3B:1–120aa.

Techniques: Immunofluorescence, Electron Microscopy, Cell Culture, Western Blot, Purification, Stable Transfection, Transfection, Staining

Journal: Autophagy

Article Title: CGAS is a micronucleophagy receptor for the clearance of micronuclei

doi: 10.1080/15548627.2021.1899440

Figure Lengend Snippet: CGAS mediated the clearance of micronuclei as an autophagy receptor. (A) Immunoblot of cell lysates and lysosomes purified from U2OS cells that had been stably transfected with HA-CGAS or HA-CGASΔ355-360 in the absence or presence of NDZ (300 nM) treatment. (B-C), Immunofluorescence assay showing the micronuclei in CGAS −/- U2OS cells that had been transfected with GFP, GFP-CGAS, GFP- CGASΔ355-360 or GFP-CGAS F357A,V360A in the absence or presence of NDZ (300 nM) treatment for 24 h followed by 48-h recovery (B). The quantification is shown in (C). (D and E) Immunofluorescence assay showing the LC3B positive micronuclei in CGAS −/- U2OS cells that had been stably transfected with GFP, GFP-CGAS, GFP- CGASΔ355-360 or GFP-CGAS F357A,V360A left untreated or treated with NDZ (300 nM) for 24 h followed by 48-h recovery (D). The quantification is shown in (E). Data are expressed as mean ± (SEM) of n = 3 independent experiments (C, E). Data are representative of n = 3 independent experiments ( A, B, D ). Nuclei were stained with DAPI (blue). Two-way ANOVA with Bonferroni post hoc test were used for statistical analysis. Scale bar: 10 μm

Article Snippet: The following primary antibodies were utilized: anti-CGAS rabbit monoclonal antibody (D1D3G; Cell Signaling Technology, 15102), anti-LC3B rabbit polyclonal antibody (MAP1LC3B:1–120aa.

Techniques: Western Blot, Purification, Stable Transfection, Transfection, Immunofluorescence, Staining