Journal: Genes

Article Title: Investigation of the Possible Role of RAD9 in Post-Diapaused Embryonic Development of the Brine Shrimp Artemia sinica

doi: 10.3390/genes10100768

Figure Lengend Snippet: Western blot analyses of As –RAD9, As –RAD1, As –HUS1, As –RAD17, and As –CHK1 at different developmental stages (0 h–3 days) of A . sinica . ( A ) The intensities of the protein bands were normalized against those of GAPDH. ( B ) Values are expressed as arbitrary units of relative value. The x-axis indicates the different protein; the y-axis shows the relative expression level. Significant differences at different development stages ( P < 0.05) were analyzed by one-way analysis of variance (ANOVA) and reported by lowercase letters (a–e).

Article Snippet: Other antibodies, such as RAD17, RAD1, CHK1, and HUS1 were purchased from BOSTER (Wuhan, China) according to sequence homology, with all homology exceeding 80%.

Techniques: Western Blot, Expressing

Journal: Genes

Article Title: Investigation of the Possible Role of RAD9 in Post-Diapaused Embryonic Development of the Brine Shrimp Artemia sinica

doi: 10.3390/genes10100768

Figure Lengend Snippet: Western blot analyses of As –RAD9, As –HUS1, As –RAD17, As –RAD1, and As –CHK1 proteins in response to temperature stresses. ( A ) The band intensity of the proteins is normalized to the band intensity of GAPDH. ( B ) The values are expressed as an arbitrary unit of relative value. Protein expression at 25 °C as control (blue), asterisk (**) indicates a statistical difference at P < 0.01, and (*) indicates 0.01 < P < 0.05.

Article Snippet: Other antibodies, such as RAD17, RAD1, CHK1, and HUS1 were purchased from BOSTER (Wuhan, China) according to sequence homology, with all homology exceeding 80%.

Techniques: Western Blot, Expressing, Control

Journal: Genes

Article Title: Investigation of the Possible Role of RAD9 in Post-Diapaused Embryonic Development of the Brine Shrimp Artemia sinica

doi: 10.3390/genes10100768

Figure Lengend Snippet: Western blot analyses of As –RAD9, As –RAD17, As –RAD1, and As –CHK1 proteins of A. sinica under different salt concentration stresses. ( A ) The band intensity of the protein is normalized to GAPDH. ( B ) The values are expressed as an arbitrary unit of relative value. The expression of the protein at a salinity of 28‰ as control (yellow), and asterisk (**) indicates a statistically significant difference of P < 0.01, and (*) indicates a 0.01 < P < 0.05.

Article Snippet: Other antibodies, such as RAD17, RAD1, CHK1, and HUS1 were purchased from BOSTER (Wuhan, China) according to sequence homology, with all homology exceeding 80%.

Techniques: Western Blot, Concentration Assay, Expressing, Control

Journal: Journal of Experimental Botany

Article Title: The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility

doi: 10.1093/jxb/erx398

Figure Lengend Snippet: Candidate SNPs (Mt4 genome version) associated with extent of symptoms and length of seedlings upon P. palmivora inoculation

Article Snippet: The M. truncatula R108 rad1 transposon insertion mutant NF-9554 (BC1-F4) was obtained from M. Harrison (Boyce Thompson Institute, Ithaca, NY, USA) and has previously been reported ( Park et al. , 2015 ).

Techniques:

Journal: Journal of Experimental Botany

Article Title: The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility

doi: 10.1093/jxb/erx398

Figure Lengend Snippet: Expression of MtRAD1 is locally induced during cortex infection with P. palmivora . (A–C) Expression levels of P. palmivora WS21 (a), M. truncatula RAD1 (b), and the P. palmivora REX3 effector candidate gene (c) at different hours post-infection (four replicates). Expression levels are calculated relative to MtUBQ (a, b) and relative to PpWS21 (c). Bars and errors bars indicate means ±SD of n =4. Comparisons made using Kruskal–Wallis and Nemenyi’s test of multiple comparisons for independent samples (Tukey). Means with different group letters are significantly different ( P <0.05) for (a) and (c). and significantly different ( P <0.01) for (b). (D) In situ hybridization using GFP or RAD1 probes on uninfected and infected M. truncatula A17 seedling root sections. GFP probes label expression of YFP-KDEL inside hyphae and sporangia of P. palmivora (black arrows). The RAD1 probe labels localized expression within cortex cells (white arrows).

Article Snippet: The M. truncatula R108 rad1 transposon insertion mutant NF-9554 (BC1-F4) was obtained from M. Harrison (Boyce Thompson Institute, Ithaca, NY, USA) and has previously been reported ( Park et al. , 2015 ).

Techniques: Expressing, Infection, In Situ Hybridization

Journal: Journal of Experimental Botany

Article Title: The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility

doi: 10.1093/jxb/erx398

Figure Lengend Snippet: Expression of hpRAD1 reduces RAD1 transcript levels as well as the degree of mycorrhization by mixed arbuscular mycorrhiza (AM). (A) Ink staining of mycorrhizal structures in hpuidA and hpRAD1 hairy roots (scale bars=200 µm); white arrowheads indicate mycorrhizal arbuscules; brightness has been enhanced in both images to increase visibility of arbuscule-filled cells. (B) Quantification of the overall degree of AM fungal colonization within root systems expressing hairpin silencing constructs targeting uidA or RAD1 . (C) Transcript levels of RAD1 and mycorrhizal symbiosis markers MtPT4 and MtBCP1 in roots expressing hpuidA and hpRAD1 constructs and grown in control conditions ( n =3) or in AM fungi mixed inoculum ( n =4). Each sample consists of five composites plants comprising at least four transformed roots, Student’s t -test was applied between constructs in each condition to compare standardized gene expression using MtUBQ as housekeeping gene and the 2 −ΔCp method (** P <0.01, *** P <0.001). Error bars show the SE.

Article Snippet: The M. truncatula R108 rad1 transposon insertion mutant NF-9554 (BC1-F4) was obtained from M. Harrison (Boyce Thompson Institute, Ithaca, NY, USA) and has previously been reported ( Park et al. , 2015 ).

Techniques: Expressing, Staining, Construct, Control, Transformation Assay, Gene Expression

Journal: Journal of Experimental Botany

Article Title: The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility

doi: 10.1093/jxb/erx398

Figure Lengend Snippet: Medicago truncatula A17 roots expressing hpRAD1 silencing constructs are impaired in colonization by P. palmivora Lili-YKDEL. (A) Overlay of maximum projections of inverted transmitted light from rhizodermis and P. palmivora Lili-YKDEL yellow fluorescence (scale bars=200 µm). (B) Maximum projection of red fluorescence from expression of nucleocytoplasmic DsRED (scale bars=200 µm). (C) Transcript levels of RAD1 and PpEF1α in roots expressing hpuidA and hpRAD1 constructs grown in control conditions (white bars, n =3) or upon infection with P. palmivora Lili-YKDEL (grey bars, n =4). Student’s t -test was applied to compare gene expression between constructs in each condition using MtUBQ as housekeeping gene and the 2 −ΔCp method (** P <0.01). Error bars show the SE.

Article Snippet: The M. truncatula R108 rad1 transposon insertion mutant NF-9554 (BC1-F4) was obtained from M. Harrison (Boyce Thompson Institute, Ithaca, NY, USA) and has previously been reported ( Park et al. , 2015 ).

Techniques: Expressing, Construct, Fluorescence, Control, Infection, Gene Expression

Journal: Journal of Experimental Botany

Article Title: The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility

doi: 10.1093/jxb/erx398

Figure Lengend Snippet: The M. truncatula rad1 mutant is impaired in colonization by P. palmivora. (A) Transcript levels of RAD1 in R108 (white bars) and rad1 (grey bars) upon infection with P. palmivora Lili-Td. (B) Transcript levels of PpEF1α in R108 (white bars) and rad1 (grey bars) upon infection with P. palmivora Lili-Td. (C) Transcript levels of PpWS21 in R108 (white bars) and rad1 (grey bars) upon infection with P. palmivora Lili-Td. For each data point, n =4 biological replicates were analysed. Student’s t -test was applied to compare gene expression between lines in each condition using MtUBQ as housekeeping gene and the 2 −ΔCp method. (D) P. palmivora progress at 24 hpi in seedling roots of R108 ( n =13) and rad1 ( n =15) measured as surface area after binary conversion (Fiji) of confocal images. (E) Confocal microscopy of M. truncatula R108 and the rad1 mutant 5 hpi with P. palmivora Lili-Td. (F) Confocal microscopy of M. truncatula R108 and the rad1 mutant 24 hpi with P. palmivora Lili-Td.(* P <0.05; ** P <0.01; *** P <0.001). Error bars show the SD. Scale bars=30 µm.

Article Snippet: The M. truncatula R108 rad1 transposon insertion mutant NF-9554 (BC1-F4) was obtained from M. Harrison (Boyce Thompson Institute, Ithaca, NY, USA) and has previously been reported ( Park et al. , 2015 ).

Techniques: Mutagenesis, Infection, Gene Expression, Confocal Microscopy

Journal: Oncogene

Article Title: THE RAD9-RAD1-HUS1 (9.1.1) COMPLEX INTERACTS WITH WRN AND IS CRUCIAL TO REGULATE ITS RESPONSE TO REPLICATION FORK STALLING

doi: 10.1038/onc.2011.468

Figure Lengend Snippet: ( A ) Depletion of RAD9 by RNAi. HeLa cells were transfected with siRNAs directed against GFP (siCtrl) or RAD9 (siRAD9) and cell lysates were prepared at the indicated time prior to immunoblotting with anti-RAD9 and anti-RAD1 antibodies. Anti-WRN antibody was used as loading control. ( B ) WRN relocalisation in HeLa cells transfected with Ctrl or RAD9 siRNAs and 48h later exposed for 6h to HU or CPT prior to immunofluorescence with anti-WRN antibody. In the panel, representative images from the untreated and the HU-treated cells are shown. Graph shows quantification of the nuclei presenting WRN focal staining under different experimental conditions. ( C ) WRN relocalisation in HeLa cells transfected with Ctrl or RAD9 siRNAs and 48h later exposed for 3h to etoposide (Etop) or bleomycin (Bleo) prior to immunofluorescence with anti-WRN antibody. In the panel, representative images from the untreated and the etoposide-treated cells are shown. Quantification of the nuclei presenting WRN focal staining after etoposide or bleomycin exposure is shown in the graph. ( D ) Depletion of WRN by RNAi. HeLa cells were transfected with siRNAs directed against GFP (siCtrl) or WRN and cell lysates prepared at the indicated time prior to immunoblotting with anti-WRN antibody. Anti-RAD9 antibody was used to verify that siWRN did not produce disruption of the 9.1.1 complex. Anti-PCNA antibody was used as loading control. ( E ) RAD9 relocalisation in nuclear foci in cells depleted of WRN. HeLa cells were transfected with Ctrl or WRN siRNAs and 48h thereafter treated for 6h with HU or CPT prior to immunofluorescence with anti-RAD9 antibody. In the panel, representative images from untreated and CPT-treated cells are shown. The percentage of nuclei showing RAD9 focal staining for each experimental condition is reported in the graph. ( F ) HeLa cells were transfected with siRNAs directed against GFP (siCtrl) or RAD9 (siRAD9) and treated with 10μM CPT for 6h, then cell lysates were prepared to immunoblotting with anti-RAD9 antibody. Anti-tubulin (Tub.) antibody was used as loading control. ( G ) WRN phosphorylation in RAD9-depleted cells. Mock and RAD9 RNAi-transfected HeLa cells were treated with 2mM HU or 10μM CPT for 6h, then cell extracts were immunoprecipitated (IP) using anti-WRN antibody. WRN phosphorylation was evaluated for the presence of a phospho-reactive band (IB) using anti-pST/Q antibodies. The total amount of WRN immunoprecipitated was determined by anti-WRN antibody (IB). Data are presented as means of three independent experiments. Error bars represent standard error.

Article Snippet: Analysis of RAD1 binding to the GST-N-WRN sub-fragments was carried out using 35 S-labelled RAD1 prepared by TnT reaction (Promega).

Techniques: Transfection, Western Blot, Control, Immunofluorescence, Staining, Disruption, Phospho-proteomics, Immunoprecipitation

Journal: Oncogene

Article Title: THE RAD9-RAD1-HUS1 (9.1.1) COMPLEX INTERACTS WITH WRN AND IS CRUCIAL TO REGULATE ITS RESPONSE TO REPLICATION FORK STALLING

doi: 10.1038/onc.2011.468

Figure Lengend Snippet: ( A ) WRN immunoprecipitates the 9.1.1 complex. HeLa cells were treated with 2mM HU or 20μM CPT for 6h, then cell lysates were immunoprecipitated (IP) using anti-WRN antibody and normal IgG as a negative control. The presence of RAD9 and RAD1 was assessed by immunoblotting (IB) using the indicated antibodies. Inputs contained 20% of the total lysates used for immunoprecipitation. (B) RAD9 immunoprecipitates WRN. HeLa cells were treated with 2mM HU or 20μM CPT for 6h. Cell extracts were immunoprecipitated (IP) with anti-RAD9 antibody and normal IgG as a negative control. The presence of WRN was evaluated by immunoblotting (IB) using the indicated antibody. ( C ) Analysis of WRN and RAD9 co-localisation after replication arrest. HeLa cells were treated with 2mM HU for 8h and subjected to immunofluorescence using mouse anti-WRN and rabbit anti-RAD9 antibodies. Representative images from HeLa cells untreated or treated with HU for 8h are presented. Insets show an enlarged portion of the nuclei for a better evaluation of the co-localisation status of WRN with RAD9 foci.

Article Snippet: Analysis of RAD1 binding to the GST-N-WRN sub-fragments was carried out using 35 S-labelled RAD1 prepared by TnT reaction (Promega).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Immunofluorescence

Journal: Oncogene

Article Title: THE RAD9-RAD1-HUS1 (9.1.1) COMPLEX INTERACTS WITH WRN AND IS CRUCIAL TO REGULATE ITS RESPONSE TO REPLICATION FORK STALLING

doi: 10.1038/onc.2011.468

Figure Lengend Snippet: ( A ) The N-terminal region of WRN interacts with RAD1. GST-tagged peptides corresponding to the N-, H-, and C- regions of the WRN protein were purified from E. coli and incubated with 5μg of HeLa nuclear extracts (NE). After separation on SDS-PAGE, the presence of RAD9 and RAD1 in the pull-down material was assessed by immunoblotting using the corresponding antibodies. The 5% of total NE was loaded as input. Coomassie Blue (CB) staining was used to show the equal input of the GST-tagged WRN fragments. ( B ) Far western analysis of the WRN interaction with the 9.1.1 complex. Recombinant 9.1.1 complex was separated using SDS-PAGE and blotted onto nitrocellulose membrane. Ponceau staining shows equal loading and transfer between the lanes ( left panel ). Single lanes were incubated with: no probe (lane 1), purified Flag-14-3-3 (lane 2) or purified Flag-WRN (lane 3) and subjected to immunoblotting using anti-Flag antibody to detect association of WRN to 9.1.1 complex ( right panel ). ( C ) Schematic representation of the N-terminal sub-fragments of WRN used to map the 9.1.1 interaction site. ( D ) Association between the different N-terminal sub-fragments of WRN and RAD1. GST-tagged peptides corresponding to the five different N-terminal sub-fragments were purified from E. coli and incubated with in-vitro-translated (IVT) 35 S-labelled RAD1. After separation on SDS-PAGE and blotting, the presence of RAD1 in the pull-down material was assessed by autoradiography. Ponceau red staining of the blot shows the amount of N-terminal sub-fragments used in the pull-down analysis. Incubation of IVT 35 S-labelled RAD1 with GST or beads alone was used as a control. Boxes indicate the identity and position of each fragment. ( E ) GST-tagged peptides corresponding to the five different N-terminal sub-fragments were purified from E. coli and incubated with 2μg of HeLa nuclear extracts. After separation on SDS-PAGE, the presence of RAD1 in the pull-down material was assessed by immunoblotting using an anti-RAD1 antibody. The 1/5 of total NE was loaded as input. Ponceau red staining of the blot shows the amount of N-terminal sub-fragments used in the pull-down analysis. Boxes indicate the identity and position of each fragment.

Article Snippet: Analysis of RAD1 binding to the GST-N-WRN sub-fragments was carried out using 35 S-labelled RAD1 prepared by TnT reaction (Promega).

Techniques: Purification, Incubation, SDS Page, Western Blot, Staining, Recombinant, Membrane, In Vitro, Autoradiography, Control

Journal: Oncogene

Article Title: THE RAD9-RAD1-HUS1 (9.1.1) COMPLEX INTERACTS WITH WRN AND IS CRUCIAL TO REGULATE ITS RESPONSE TO REPLICATION FORK STALLING

doi: 10.1038/onc.2011.468

Figure Lengend Snippet: ( A ) Western blotting on extracts from WS cells stably expressing the Flag-tagged wild-type WRN (WRN wt ) or the 112-121 deletion mutant (WRN del ) showing levels of WRN using an anti-WRN antibody. WS cells were used as a negative control and tubulin as loading control. ( B ) Analysis of the association with 9.1.1 of the WRN mutant protein with deletion in the 9.1.1-binding region. Five μg of wild-type or 112-121 deletion mutant GST-tagged N-terminal fragment of WRN (1-550) was purified from E. coli and incubated with 2μg of HeLa nuclear extracts. After release in sample buffer and separation on SDS-PAGE, the presence of RAD1 in the pull-down material was assessed by immunoblotting using an anti-RAD1 antibody. One-tenth of the released material was subjected to immunoblotting using an anti-GST antibody to visualize the amount of GST-NWRN fragments. Pull-down using uncoupled GST-binding beads was used as negative control. ( C ) Analysis of WRN relocalisation to nuclear foci after replication arrest. Images show WRN nuclear distribution with or without an 8h HU treatment. The inset shows the percentage of WRN positive nuclei. Data are presented as means of three independent experiments+/− standard errors. ( D ) Analysis of WRN-9.1.1 association in cells expressing WRN del . 293T cells transiently expressing the Flag-tagged WRN wt or the Flag-tagged WRN del protein were treated with 2mM HU for 8h prior to lysis and immunoprecipitation using anti-Flag antibody. The presence of RAD9 and RAD1 were assessed by immunoblotting (IB) using the indicated antibodies. Immunoprecipitation using normal IgG was used as a negative control. Inputs contained 15% of the total lysates used for immunoprecipitation. A fraction of the lysate (1/50) was also analysed by immunoblotting to evaluate the amount of the wild-type and mutant form of WRN expressed in 293T cells. RAD9 immunoblotting was used to confirm the presence of RAD9 in the lysates and as loading control. ( E ) Analysis of WRN phosphorylation at S/TQ sites in the 112-121 deletion WRN mutant after HU treatment. Cells expressing the wild-type and the WRN del mutant were treated with 2mM HU for 6h prior to lysis and immunoprecipitation using an anti-Flag antibody. WRN phosphorylation was evaluated by immunoblotting in the WRN immunoprecipitates with an anti-pST/Q antibody (IB: pS/TQ). The total amount of the immunoprecipitated WRN protein was determined by anti-WRN immunobloting (IB: WRN). Immunoprecipitation using normal mouse IgG (IgG) was used as a negative control.

Article Snippet: Analysis of RAD1 binding to the GST-N-WRN sub-fragments was carried out using 35 S-labelled RAD1 prepared by TnT reaction (Promega).

Techniques: Western Blot, Stable Transfection, Expressing, Mutagenesis, Negative Control, Control, Binding Assay, Purification, Incubation, SDS Page, Lysis, Immunoprecipitation, Phospho-proteomics