erap1 construct  (Addgene inc)

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: Erap1 (a, c, e) and Tap1 (b, d, e) expression in normal neural stem cells (NSCs, n=3), MP tumors (n=3), MG tumors (n=3), MG tumors overexpressing DNp53 (MG+P) (n=3) and MP tumors overexpressing GFI1 (MP+G) (n=3) were measured by qRT-PCR (a-d) and by western blotting (e); quantification of 3 independent experiments is shown below the western blot. Error bars represent means ± SD. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. p-values were determined by two-sided unpaired t-test. In (a), NS: p=0.21 (MG vs NSC), ** p=0.0019 (MG vs MP). In (b) NS, p=0.33 (MG vs NSC); *** p=1.9E-05. In (c), *** p=1E-04 (MP vs MG) and p=8E-05 (MG vs MG+P). In (d), *** p=2.3E-05 (MP vs MG), ** p=0.003 (MG vs MG+P, Tap1). In (e), * refers to p=0.002 (MP vs MG, Erap1), p=0.0052 (MG vs MG+P,Erap1), p=0.0097 (MP vs MG, Tap1), p=0.0049 (MG vs MG+P, Tap1). (f-g) Analysis of ERAP1 (f) and TAP1 (g) mRNA levels in human Sonic Hedgehog-associated MB with mutant (red) or wild-type (WT, blue)) TP53. Fragments Per Kilobase per Million mapped reads (FPKM) of ERAP1 and TAP1 are shown. p-values were determined by two-sided Wilcoxon rank sum test. Boxplot center lines show median, box limits indicate the 25th and 75th percentiles, lower and upper whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles, respectively. (h) Putative p53 binding sites in the mouse Erap1 and Tap1 promoters. (i-j) Chromatin Immunoprecipitation (ChIP) and PCR analysis of p53 binding site-containing regions in the Erap1 (i) and Tap1 (j) promoters. qPCR results for anti-p53 ChIP (IP p53) or isotype control ChIP (IP Ctl) in MG (n=3) and MP (n=3) tumor cells. p-values were determined by two-sided unpaired t-test * p=0.0046 (Erap1) and ** p=0.0011 (Tap1). (k) Putative p53 binding sites in the human ERAP1 and TAP1 promoters. (l-m) ChIP and PCR analysis of the p53 binding site-containing regions in the ERAP1 (l) and TAP1 (m) promoters. qPCR results for anti-p53 ChIP (IP p53) or isotype control ChIP (IP Ctl) in p53 mutant PDXs (RCMB18, Icb984, BT084) and p53 wild-type PDXs (Icb1299, RCMB40) (n=3 for each tumor type). p-values were determined by two-sided unpaired t-test. * p=0.026 (Icb1299, ERAP1), p=0.01 (Icb1299, TAP1), p=0.002 (RCMB40, TAP1) ; ** p=0.001 (RCMB40, ERAP1).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Control

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: MG (a, c, e, g) and MP (b, d, f, h) tumor cells were treated in vitro for 48h with IFNα (20 ng/mL), IFNγ (20 ng/mL), TNF (50 pg/mL) or LtβRag (1.6μg/mL). MHC-I expression in untreated cells (Ctl, black histograms) and cells treated with IFNα (red, a, b), IFNγ (red, c, d), TNF (green, e, f) or LTβRag (blue, g, h) were analyzed by FACS. Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below each histogram; data points represent MFIs for individual tumor samples. p-values were determined by two-sided unpaired t-test. p-values are indicated on the corresponding graphs. Erap1 (i) and Tap1 (j) mRNA expression in untreated MP tumor cells (Ctl) or MP tumor cells treated with TNF, LTβRag or IFNγ were determined by qRT-PCR (n=3). Data points represent expression values for individual tumor samples; error bars represent the mean ± SD. p-values were determined by two-sided unpaired t-test. In (i), NS, p=0.351;* p=0.006, ***p=0.0004. In (j), NS, p=0.453, * p=0.0086, ** p=0.003. (k) Tap1 and Erap1 protein levels were assayed by western blotting. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. Relative protein levels of Erap1 (l) and Tap1 (m) normalized to actin are shown for three independent samples. Error bars represent mean ± SD; data points represent expression values for individual tumor samples. p-values were determined by two-sided unpaired t-test. In (l), NS p=0.133, * p=0.0025, ** p=0.0004 ; In (m), NS = 0.378, * p=0.029, ** p=0.0116.

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: In Vitro, Expressing, Fluorescence, Quantitative RT-PCR, Western Blot

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: (a-d) MG tumor cells were transduced with control shRNA (shCtl) or shRNAs targeting Erap1 (shErap1#1, shErap1#2). Knockdown efficiency was determined by western blotting in three independent tumor samples (a). MHC-I expression was determined by FACS in control cells (shCtl, black) and Erap1 knockdown cells (shErap1, red). Western blots are cropped at the molecular weights for Erap1 (120 kDa) and β-actin (42 kDa); original blots are available in Source Data. (b). Quantification of the mean fluorescence intensity (MFI) values for three independent tumors is shown below each histogram; data points represent MFIs for individual tumor samples. p-values, determined by two-sided unpaired t-test, are indicated on each bar graph. (c-d) Erap1 knockdown cells were transplanted into aB6 mice. Bioluminescence imaging of representative mice (c) and survival curves (n=6) (d) are shown. p-values for the difference in survival between shErap1 and shCtl were determined using the two-sided log-rank (Mantel-Cox) test. * p=0.024; ** p= 0.0075. (e-i) MP tumor cells were transduced with empty vector (vect) or vectors encoding Erap1, Tap1 or both. Erap1 (e) and Tap1 (f) expression levels were assessed by western blotting in three independent tumor samples. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. (g) Analysis of MHC-I expression by FACS in control cells (vect, black) and Erap1 + Tap1 overexpressing cells (pink). Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below the histogram; data points represent MFIs for individual tumor samples. The p-value was determined by two-sided unpaired t-test. (h, i) MP tumor cells transduced with control vector (vect, black), Tap1 (green), Erap1 (blue) or Erap1 + Tap1 (pink) were orthotopically transplanted into aB6 mice. Bioluminescence imaging of representative mice (h) and survival curves (n=6 per group) (i) are shown. p-values were determined by the two-sided log-rank (Mantel-Cox) test. NS (Ctl vs. Tap1), p=0.79; * (Ctl vs. Erap1) p=0.0047; ** (Ctl vs. Erap1+Tap1) p=0.0005. Mice carrying MP tumors expressing Erap1 + Tap1 survive significantly longer than mice carrying tumors expressing Erap1 alone (p=0.0016 for Erap1 vs. Erap1 + Tap1) or Tap1 alone (p = 0.0005 for Tap1 vs. Erap1 + Tap1).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Transduction, Control, shRNA, Knockdown, Western Blot, Expressing, Fluorescence, Imaging, Plasmid Preparation

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: (a, b) FACS analysis of MHC-I expression in MP tumor cells that were untreated (Ctl, black histograms) or treated for 48h with TNFR1 agonist (0.5μg/mL, purple histogram, a) or TNFR2 agonist (1.67nM, red histogram, b). (c) MP tumor cells generated from TNFR2 knockout (TNFR2 KO) mice were treated for 48h with no stimulus (Ctl, black histogram) or with TNF (50 pg/mL, green histogram) and then analyzed by FACS. Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below each histogram; data points represent MFIs for individual tumor samples. p-values were determined by two-sided unpaired t-test. p-values are indicated on the corresponding graphs. (d-f) MP tumor cells were untreated (NT) or treated with TNF for 15’, 30’ or 1h. Expression of RelA and RelB protein were assessed by western blotting in nuclear extracts (d) and in total cellular protein extracts (e). Histone H3 and GAPDH were used as controls for nuclear and total extracts respectively. Western blots are cropped at the molecular weights for RelA (65 kDa), RelB (68kDa), Histone H3 (17kDa) and GAPDH (37 kDa); original blots are available in Source Data. (f) Quantification of Western blots for RelA (black) and RelB (blue) protein levels in nucleus relative to levels in total extract at 0’, 15’, 30’ and 60’ for three independent experiments. p-values were determined by two-sided unpaired t-test. For RelA, * p=0.049 (15’) ; ***p=0.0032 (30’) ; **p=0.0043 (1h); for RelB: NS, p=0.088 (15’) ; *p=0.037 (30’) ; NS p=0.073 (1h). (g, i) Putative binding sites for the RelA-p50 heterodimer in the mouse Erap1 and Tap1 promoters. (h, j) MP tumor cells were treated for 1h with no stimulus (ctl), TNF or LtβRag. Chromatin Immunoprecipitation (ChIP) was performed using antibodies specific for RelA (h) or p50 (j), and precipitates were analyzed by qPCR for the presence of the RelA-p50 binding site-containing regions of the Erap1 and Tap1 promoters. Data represent the mean fold-induction ± SD. p values were determined by two-sided unpaired t test. In (h), * p=0.0034, **p=0.003, ***p=2.6E-06 (TNF, Erap1), p=1E-04 (TNF, Tap1). (k) MP tumor cells were transduced with control shRNA (shCtl) or shRNAs targeting RelA (shRelA#832, shRelA#833), p50 (shp50#484, shp50#485) or RelB (shRelB#494, shRelB#495). MHC-I expression was determined by FACS in control cells (shCtl) and RelA, p50 or RelB knockdown cells treated in vitro for 48h with vehicle (DMSO, ctl), TNF or LtβRag. Data shown represent quantification of mean fluorescence intensity (MFI) for three independent experiments; data points represent MFIs for individual tumor samples. p values were determined by two-sided unpaired t test. shCtl : ** p=0.0019, *** p=1.1E-05; shRelA (#832): NS, Not significant p=0.063 (TNF) and p=0.098 (LtβRag); shRelA (#833): *p=0.035, **p=0.009; shp50 (#484): NS, Not significant p=0.341 (TNF), p=0.125 (LtβRag); shp50 (#485): NS, p=0.601, *p=0.049 ; shRelB (#494) *** p=1.8E-05 (TNF), p=2.3E-04 (LtβRag); shRelB (#495): *** p=9E-06 (TNF), p=1.6E-05 (LtβRag).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Expressing, Generated, Knock-Out, Fluorescence, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Transduction, Control, shRNA, Knockdown, In Vitro

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Highly heterogeneous constitutive and IFN-γ-inducible ERAP1/ERAP2 expression in melanoma cells. a The constitutive mRNA expression levels of ERAP1 and ERAP2 in primary melanocytes and representative members of the panel of melanoma cell lines as indicated on the x-axis were determined by qRT-PCR using ERAP1- and ERAP2-specific primer sets as described in “Materials and methods” and supplementary Table 1. The relative mRNA expression levels were normalized to GAPDH serving as a control. In addition, the ERAP/GAPDH ratio of the primary melanocytes were set to 1. For the determination of IFN-γ inducibility of ERAP at the transcriptional and translational level, representative qRT-PCR (b) and Western blot analyses (c) were performed with a set of four selected melanoma cell lines (Buf1287, Colo857, FM6 and FM81), which were either left untreated or treated with IFN-γ (48 h) as described in “Materials and methods”. Western blots were performed using the set of murine anti-ERAP1-, anti-ERAP2- and β-actin-specific antibodies. In addition, immunostainings targeting HLA class I HC served as positive controls for the IFN-γ treatment

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Effects of ERAP siRNA transfection on the relative ERAP1 mRNA and the corresponding HLA class I surface expression levels. The siRNA-mediated reduction of ERAP1 expression was monitored in a panel of melanoma cell lines (UKRV, Buf1287 and Buf1379) by qRT-PCR (a). The results are expressed as ratios of ERAP1 mRNA expression levels normalized to GAPDH mRNA expression levels as described in “Materials and methods”. The corresponding expression levels determined with the non-targeting (nt) control siRNA were set to 100%, respectively. All ERAP1-targeting siRNA transfectants show decreased ERAP1 transcription rates. b The HLA class I surface antigen expression was determined by flow cytometry as described in “Materials and methods” and is represented as the mean fluorescence intensity (MFI), the HLA class I surface expression of cells transfected with the non-sense control siRNA (nt) was set as 100%. The siRNA-mediated reduction of the ERAP1 mRNA levels caused slightly increased HLA class I expression levels in each of the analysed cell lines. The cell lines correspond to the one in a

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Transfection, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Heterogeneous ERAP promoter activity in melanoma cells. a The wt ERAP1 promoter and the pGL3 enhancer vector, which served as a control, were transiently transfected into a series of melanoma cells as indicated on the x-axis. Cells were left untreated or treated for 24 h with IFN-γ before the given ERAP1 (a) promoter activities were determined as described in “Materials and methods”. All results are expressed as relative luciferase activity (RLU) normalized to the corresponding β-galactosidase activity. Grey bars represent untreated cells; black bars IFN-γ-treated cells. b The relative activities of the wt ERAP1 (E1_WT) and mutated ERAP1 promoter (E1_1066) constructs as well as of the corresponding mock control (pGL3 enhancer vector) were transfected in the melanoma cell line Colo857 and the keratinocyte cell line HaCaT serving as a control. The relative promoter activities are expressed as RLU as outlined above in a. The mutation located at nt position 1066 leads to a markedly reduced transcriptional activity, independent from the cellular background. c Altered aminopeptidase activities of ERAP1 variants. The relative enzymatic activities of various ERAP1 constructs in the microsome fractions were determined as described in “Materials and methods”. The ERAP1-negative cell line Buf1182 was either transfected with the empty vector (mock), the wtERAP1 (E1_WT), the novel mut349ERAP1 (E1_349) or with the previously described enzymatically almost inactive ERAP1 variant mut320ERAP1 (E1_320). The relative enzymatic activities are expressed as RLU normalized to the relative ERAP1 enrichment within the indicated microsomal fractions as determined by ERAP1-targeting Western blot analyses. The newly characterized variant with the amino acid substitution M→V at position 349, which is located near the active site, slightly enhances the enzymatic activity of ERAP1

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Activity Assay, Plasmid Preparation, Control, Transfection, Luciferase, Construct, Mutagenesis, Variant Assay, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Heterogeneous mRNA and/or protein expression of ER-resident aminopeptidases, IRF1 and HLA class I in human melanoma cell lines

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: SNPs within the ERAP1 coding region

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: