p53r280k  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Intrinsic adriamycin resistance in p53-mutated breast cancer is related to the miR-30c/FANCF/REV1-mediated DNA damage response

doi: 10.1038/s41419-019-1871-z

Figure Lengend Snippet: a qRT-PCR and b Western blot analyses showing the mRNA and protein expression of REV1 and FANCF in untreated MCF-7 and MDA-MB-231 cells (blank), as well as cells transfected with miR-30a, miR-30b, miR-30c, miR-30d, miR-30e mimic and negative control (NC, scrambled control oligonucleotides). ** p < 0.01 vs. NC group. c The REV1 and FANCF 3′UTR binding sites of miR-30c and the REV1 and FANCF 3′UTR mutation sites in the mutated luciferase plasmid. d Dual-luciferase activity in 293 T cells transfected with miR-30c mimic (20 nM), NC (20 nM) and a wild-type or mutated 3′UTR of REV1 or FANCF, ** p < 0.01 vs. NC group. NS indicates no significant difference. NC: negative control, scrambled control oligonucleotides. e Western blot analysis showing the protein expression of REV1, FANCF, and FANCD2 in MCF-7 and MDA-MB-231 cells transfected with miR-30c inhibitors (20 nM) or the NC inhibitor (20 nM) and untreated MCF-7 and MDA-MB-231 cells (Blank). The graph showing the statistical results is located on the right. * p < 0.05, ** p < 0.01 vs. Inh-NC group. Data represent the mean ± SD of three independent experiments

Article Snippet: The BrCa cell lines MCF-7, ZR-75-1, T-47D, MDA-MB-231 (ATCC, USA) and MCF-10A (ATCC, USA) were cultured under standard conditions, as recommended by their manufacturers. pMKO.1 puro p53 shRNA (Plasmid #10671) and pCMV-Neo-Bam p53wt (Plasmid #16434) were obtained from Addgene ( http://www.addgene.org ). p53R280K cDNA, REV1 cDNA and FANCF cDNA plasmids without 3′ UTR region were obtained from GeneChem ( www.genechem.com.cn ).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Negative Control, Control, Binding Assay, Mutagenesis, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell Death & Disease

Article Title: Intrinsic adriamycin resistance in p53-mutated breast cancer is related to the miR-30c/FANCF/REV1-mediated DNA damage response

doi: 10.1038/s41419-019-1871-z

Figure Lengend Snippet: a Real-time PCR analysis of miR-30c basal expression in wtp53 (MCF-10A, MCF-7, and ZR-75-1) and mutp53 (MDA-MB-231, and T-47D) BrCa cells. * p < 0.05, wtp53 vs. mutp53 BrCa. b wtp53 and mutp53 BrCa cells were treated with ADR (0.5 µM) for 48 h. The expression of miR-30c was evaluated by qRT-PCR. * p < 0.05,** p < 0.01 vs. control group. NS indicates no significant difference. c MDA-MB-231 and T47D cells were transiently transfected with the NC or miR-30c mimic and were treated with various ADR concentrations. The relative viability of the MDA-MB-231 and T47D cells was determined via CCK-8 assays ( n = 3). * p < 0.05 vs. NC group. d The relative viability of the MDA-MB-231and T47D cells was detected by CCK-8 assays 48 h after transfection with NC (20 nM), miR-30c mimic (20 nM), FANCF (4 µg), REV1 cDNA(4 µg) or FANCF and REV1 cDNA. FANCF and REV1 constructs are plasmids without 3′ UTR region. # p < 0.05 vs. miR-30c group, * p < 0.05 vs. NC group. e MCF-7 and MDA-MB-231 cells were injected subcutaneously into nude mice. A cholesterol-conjugated miR-30c agomir or negative control (NC) agomir was intratumorally injected 7 days after tumor formation, and agomir and ADR were intratumorally injected every two days ( n = 5). f Xenograft tumor growth was monitored. # p < 0.05, ## p < 0.01 vs. ADR group; * p < 0.05, ** p < 0.01 vs. NC(-) agomir group. g Tumor weight at sacrifice. * p < 0.05, *** p < 0.001 vs. NC(−) agomir group; # p < 0.05, ### p < 0.001 vs. ADR group. Data represent the mean ± SD ( n = 5, each group). h Photographs of the removed tumors at day 34 after the implantation. i Western blot analysis for FANCF and REV1 in the MCF-7 and MDA-MB-231 xenografts after treatment with the cholesterol-conjugated miR-30c agomir or the NC agomir. ** p < 0.01 vs. NC group

Article Snippet: The BrCa cell lines MCF-7, ZR-75-1, T-47D, MDA-MB-231 (ATCC, USA) and MCF-10A (ATCC, USA) were cultured under standard conditions, as recommended by their manufacturers. pMKO.1 puro p53 shRNA (Plasmid #10671) and pCMV-Neo-Bam p53wt (Plasmid #16434) were obtained from Addgene ( http://www.addgene.org ). p53R280K cDNA, REV1 cDNA and FANCF cDNA plasmids without 3′ UTR region were obtained from GeneChem ( www.genechem.com.cn ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Control, Transfection, CCK-8 Assay, Construct, Injection, Negative Control, Western Blot

Journal: Cell Death & Disease

Article Title: Intrinsic adriamycin resistance in p53-mutated breast cancer is related to the miR-30c/FANCF/REV1-mediated DNA damage response

doi: 10.1038/s41419-019-1871-z

Figure Lengend Snippet: a Schematic representation of NFYC (miR-30c host gene) and putative p53 binding sites in intron 5 of NFYC . b HEK-293T cells were treated with pcDNA3.1-p53 (wtp53), pcDNA3.1-p53R280K (mutp53), ADR (0.5 µM), pifithrin-α (10 µM), pifithrin-α + ADR or the miR-30c promoter constructs (in the pGL3 vector). Relative luciferase activity was assayed. ** p < 0.01 vs. PGL3 vector, ## p < 0.01 vs. pifithrin-α group. c ChIP assay showing endogenous p53 bound to the miR-30c promoter in the p53 binding site region in MCF-7 cells. d MCF-7 and MDA-MB-231 cells were treated with ADR (0.5 µM) for 12, 24, and 48 h. The expression of mature miR-30c and pri-miR-30c was evaluated by qRT-PCR. The protein expression of p-p53, p53, and p21 was examined by Western blotting. * p < 0.05, ** p < 0.01 vs. 0 h group. e MCF-7 and MDA-MB-231 cells were treated with control, p53shRNA, ADR, or p53shRNA + ADR for 48 h. qRT-PCR showed that the miR-30c expression changes. The protein expression of p53 was examined by Western blotting. f MCF-7 and MDA-MB-231 cells were transfected with NC inhibitor (20 nM) or miR-30c inhibitor (20 nM) and were then treated with ADR and p53cDNA. The expression of FANCF and REV1 was analyzed by Western blotting. MCF-7 cells: * p < 0.05, ** p < 0.01 vs. Blank group; # p < 0.05 vs. ADR group, ## p < 0.01, vs. p53cDNA group. MDA-MD-231 cells: * p < 0.05 vs. Blank group; # p < 0.05 vs. p53cDNA group. g FANCF and REV1 expression in MCF-7 and MDA-MB-231 cells transfected with p53 shRNA, miR-30c mimic (miR-30c) or the negative control (NC). The expression of FANCF and REV1 was analyzed by Western blotting. ** p < 0.01, *** p < 0.001 vs. Blank group; # p < 0.05 vs. Blank group. h qRT-PCR analysis of miR-30c expression in ADR-treated MCF-7 and MDA-MB-231 xenografts. Western blot analysis of p-p53, p53, FANCF, and REV1 expression in ADR-treated MCF-7 and MDA-MB-231 xenografts. ** p < 0.01, *** p < 0.001 vs. Blank group. NS indicates no significant difference. Data represent the mean ± SD ( n = 3, each group)

Article Snippet: The BrCa cell lines MCF-7, ZR-75-1, T-47D, MDA-MB-231 (ATCC, USA) and MCF-10A (ATCC, USA) were cultured under standard conditions, as recommended by their manufacturers. pMKO.1 puro p53 shRNA (Plasmid #10671) and pCMV-Neo-Bam p53wt (Plasmid #16434) were obtained from Addgene ( http://www.addgene.org ). p53R280K cDNA, REV1 cDNA and FANCF cDNA plasmids without 3′ UTR region were obtained from GeneChem ( www.genechem.com.cn ).

Techniques: Binding Assay, Construct, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Control, Transfection, shRNA, Negative Control

Journal: Cell Death & Disease

Article Title: Intrinsic adriamycin resistance in p53-mutated breast cancer is related to the miR-30c/FANCF/REV1-mediated DNA damage response

doi: 10.1038/s41419-019-1871-z

Figure Lengend Snippet: a miR-30c expression in normal breast tissues ( n = 113) and BrCa tissues ( n = 1109) in The Cancer Genome Atlas (TCGA) dataset. The p value was calculated via a nonparametric Mann-Whitney test. b Kaplan–Meier overall survival curves according to miR-30c expression in METABRIC BrCa patient cohorts. c A whisker plot showing miR-30c expression in patients with mutp53 tumors ( n = 285) and wtp53 tumors (n = 775) using TCGA database. d Representative images of miR-30c in situ hybridization and FANCF and REV1 IHC for 118 cases of BrCa expressing wtp53 and mutp53. Magnification, ×200. Small frames indicate the magnified regions. e Quantitative data for miR-30c, FANCF and REV1 protein staining in d . Statistical significance was determined by Wilcoxon rank-sum tests. f Correlation analysis of miR-30c and REV1 or FANCF protein expression in BrCa patients

Article Snippet: The BrCa cell lines MCF-7, ZR-75-1, T-47D, MDA-MB-231 (ATCC, USA) and MCF-10A (ATCC, USA) were cultured under standard conditions, as recommended by their manufacturers. pMKO.1 puro p53 shRNA (Plasmid #10671) and pCMV-Neo-Bam p53wt (Plasmid #16434) were obtained from Addgene ( http://www.addgene.org ). p53R280K cDNA, REV1 cDNA and FANCF cDNA plasmids without 3′ UTR region were obtained from GeneChem ( www.genechem.com.cn ).

Techniques: Expressing, MANN-WHITNEY, Whisker Assay, In Situ Hybridization, Staining