Journal: G3: Genes | Genomes | Genetics

Article Title: Regulation of Drosophila brain development and organ growth by the Minibrain/Rala signaling network

doi: 10.1093/g3journal/jkae219

Figure Lengend Snippet: Mnb interacts with Reps and Rlip. a) Network clusters identified in Mnb AP-MS include known Mnb interactors Wap and Reph (left cluster) and a highly interconnected group of endocytosis related proteins (right cluster). b-g) Co-immunoprecipitation (co-IP) experiments in Drosophila S2 cells showing the binding interactions among Mnb, Reps, and Rlip using the indicated protein combinations and IP directions. Mnb showed pairwise interactions with Reps (b, c) and Rlip (d, e), and a ternary interaction with Reps and Rlip (f, g). h) Phos-tag analysis of Reps phosphorylation by Mnb. Bottom: regular SDS/PAGE gel. Mnb-KR, a kinase dead version of Mnb (Mnb K193R mutant) ( Degoutin et al. 2013 ). i) Co-IP of DYRK1A and REPS1/2 in HEK293T cells through IP of REPS1/2. IP: immunoprecipitation, IB: immunoblot.

Article Snippet: pMT-Reps-Flag and pMT-Reps-HA were generated by cloning Reps-RA isoform from FMO04419 (DGRC) into pMT-V5-His vector (Invitrogen). pMT-Mnb-V5 was generated by cloning Mnb-RH isoform from FMO12028 (DGRC) into pMT-V5-His vector. pMT-Rlip-HA was generated by cloning the Rlip-RA isoform from GH01995 (DGRC) into pMT-V5-His vector. pMT-mCherry-Rlip and pMT-Rlip-mCherry were cloned from pMT-Rlip-HA using NEB HiFi Assembly kit (NEB) to exclude the retained intron and incorporate the mCherry open reading frame into pMT-V5-His vector. pcDNA3.1-DYRK1A-V5 was generated by cloning DYRK1A from pMH-SFB-DYRK1A (Addgene) into pcDNA3.1(−) (ThermoFisher). pcDNA3.1-REPS2-Flag was obtained from Sinobiological. pcDNA3.1-REPS1-Flag was generated by cloning REPS1 from pcMV-REPS1 (Sinobiological) into pcDNA3.1(−).

Techniques: Protein-Protein interactions, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Phospho-proteomics, SDS Page, Mutagenesis, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Quantitative RT-PCR, Comparison, Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Control, Comparison, Binding Assay, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection, Negative Control, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Western Blot, Transfection, Incubation, Control, Flow Cytometry, Expressing, CCK-8 Assay, Comparison, Cell Culture, Staining, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Staining, Transfection, Control, Immunostaining