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Arngården, Linda
Publications (10 of 12) Show all publications
Klaesson, A., Grannas, K., Ebai, T., Heldin, J., Koos, B., Leino, M., . . . Landegren, U. (2018). Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes. Scientific Reports, 8, Article ID 5400.
Open this publication in new window or tab >>Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5400Article in journal (Refereed) Published
Abstract [en]

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-340077 (URN)10.1038/s41598-018-23582-1 (DOI)000428618900043 ()29599435 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568 264737 294409Swedish Foundation for Strategic Research Swedish Research Council
Note

Ola Söderberg and Ulf Landegren jointly supervised this work

Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-08-06Bibliographically approved
Löf, L., Arngården, L., Olsson-Strömberg, U., Siart, B., Jansson, M., Dahlin, J. S., . . . Kamali-Moghaddam, M. (2017). Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia. Scientific Reports, 7, 1-9, Article ID 623.
Open this publication in new window or tab >>Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-9, article id 623Article in journal (Refereed) Published
Abstract [en]

Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-319726 (URN)10.1038/s41598-017-00755-y (DOI)000398162400034 ()28377570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 294409Swedish Cancer SocietySwedish Research Council
Available from: 2017-04-07 Created: 2017-04-07 Last updated: 2017-05-15Bibliographically approved
Arngården, L. (2016). Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A cell that senses signals from its environment uses proteins for signal transduction via post translational modifications (PTMs) and protein- protein interactions (PPIs) from cell membrane into the nucleus where genes controlling cell proliferation, differentiation and apoptosis can be turned on or off, i.e. changing the phenotype or fate of the cell. Aberrations within such proteins are prone to cause diseases, such as cancer. Therefore, it is important so study aberrant signaling to be able to understand and treat diseases.

In this thesis, signaling aberrations of PTMs and PPIs were analyzed with the use of the in situ proximity ligation assay (in situ PLA), and the thesis also contain method development of rolling circle amplification (RCA), which is the method used for signal amplification of in situ PLA reaction products.

Paper I considers the integrity of RCA products. Here, the aim was to generate a smaller and more compact RCA product, for more accurate either visual or automated analysis. This was achieved with the use of an additional so called compaction oligonucleotide that during RCA was able to bind and pull segments of RCA products closer together. The compaction oligonucleotide served to increase the signal to noise ratio and decrease the number of false positive signals.

The crosstalk between the Hippo and TGFβ signaling pathways were studied in paper II. Activity of the Hippo signaling pathway is regulated by cell density sensing and tissue control. We found differences in amounts and localization of interactions between the effector proteins of the two pathways depending on cell density and TGFβ stimulation.

In paper III the NF-кB signaling pathway constitutively activated in chronic lymphocytic leukemia (CLL) was studied. A 4 base-pair frameshift deletion within the NFKBIE gene, which encodes the negative regulator IкBε, was found among 13 of a total 315 cases by the use of targeted deep sequencing. We found reduced levels of IкBε protein, decreased p65 inhibition, and increased phosphorylation, along with increased nuclear localization of p65 in NFKBIE deleted cases compared to healthy cases.

Crosstalk between the Hippo and Wnt signaling pathway are studied within paper IV. Here, we found differences in cellular localization of TAZ/β-catenin interactions depending on colon cancer tumor stage and by further investigate Hippo/WNT crosstalk in cell line model systems we found an increase of complex formations involved in the crosstalk in sparse growing HEK293 cells compared to dense growing cells. Also, active WNT3a signaling was affected by cell density. Since cell density showed to have a big effect on Hippo/WNT crosstalk we continued to investigated the effect of E-cadherin, which has a function in cell junctions and maintenance of epithelial integrity on Hippo/WNT crosstalk. Interestingly, we found that E-cadherin is likely to regulate Hippo/WNT crosstalk.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 45
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1202
Keywords
cell signaling, Wnt, Hippo, TGFB
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-281716 (URN)978-91-554-9529-9 (ISBN)
Public defence
2016-05-20, BMC, B41, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-04-28 Created: 2016-03-29 Last updated: 2016-05-12
Clausson, C.-M., Arngården, L., Ishaq, O., Klaesson, A., Kühnemund, M., Grannas, K., . . . Söderberg, O. (2015). Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio. Scientific Reports, 5, 12317:1-10, Article ID 12317.
Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, p. 12317:1-10, article id 12317Article in journal (Refereed) Published
National Category
Medical Image Processing
Research subject
Computerized Image Processing
Identifiers
urn:nbn:se:uu:diva-260286 (URN)10.1038/srep12317 (DOI)000358358900001 ()26202090 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 259796Swedish Research Council
Available from: 2015-07-23 Created: 2015-08-18 Last updated: 2018-02-27Bibliographically approved
Grannas, K., Arngården, L., Lönn, P., Mazurkiewicz, M., Blokzij, A., Zieba Wicher, A. & Söderberg, O. (2015). Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes. Journal of Molecular Biology, 427(21), 3407-3415
Open this publication in new window or tab >>Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
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2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 21, p. 3407-3415Article in journal (Refereed) Published
Abstract [en]

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-268434 (URN)10.1016/j.jmb.2015.04.015 (DOI)000363823200006 ()25937570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568Swedish Research Council
Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2017-12-01Bibliographically approved
Mansouri, L., Sutton, L.-A., Ljungström, V., Bondza, S., Arngården, L., Bhoi, S., . . . Rosenquist Brandell, R. (2015). Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia. Journal of Experimental Medicine, 212(6), 833-843
Open this publication in new window or tab >>Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia
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2015 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 212, no 6, p. 833-843Article in journal (Refereed) Published
Abstract [en]

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-279237 (URN)10.1084/jem.20142009 (DOI)000355569300001 ()25987724 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), b2011080Swedish Cancer SocietySwedish Research CouncilNIH (National Institute of Health), CA81554; CA081554EU, European Research Council, 259796EU, FP7, Seventh Framework Programme, 306242
Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2018-01-10Bibliographically approved
Fristedt Duvefelt, C., Lub, S., Prasoon, A., Arngården, L., Hammarberg, A., Maes, K., . . . Jernberg-Wiklund, H. (2015). Increased resistance to proteaome inhibitors in multiple myeloma mediated by cIAP2: implications for a combinatorial treatment. OncoTarget, 6(24), 20621-20635
Open this publication in new window or tab >>Increased resistance to proteaome inhibitors in multiple myeloma mediated by cIAP2: implications for a combinatorial treatment
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2015 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 6, no 24, p. 20621-20635Article in journal (Refereed) Published
Abstract [en]

Despite the introduction of new treatment options for multiple myeloma (MM), a majority of patients relapse due to the development of resistance. Unraveling new mechanisms underlying resistance could lead to identification of possible targets for combinatorial treatment. Using TRAF3 deleted/mutated MM cell lines, we evaluated the role of the cellular inhibitor of apoptosis 2 (cIAP2) in drug resistance and uncovered the plausible mechanisms underlying this resistance and possible strategies to overcome this by combinatorial treatment. In MM, cIAP2 is part of the gene signature of aberrant NF-kappa B signaling and is heterogeneously expressed amongst MM patients. In cIAP2 overexpressing cells a decreased sensitivity to the proteasome inhibitors bortezomib, MG132 and carfilzomib was observed. Gene expression analysis revealed that 440 genes were differentially expressed due to cIAP2 overexpression. Importantly, the data imply that cIAPs are rational targets for combinatorial treatment in the population of MM with deleted/mutated TRAF3. Indeed, we found that treatment with the IAP inhibitor AT-406 enhanced the anti-MM effect of bortezomib in the investigated cell lines. Taken together, our results show that cIAP2 is an important factor mediating bortezomib resistance in MM cells harboring TRAF3 deletion/mutation and therefore should be considered as a target for combinatorial treatment.

National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-242568 (URN)000360138200076 ()
Funder
Swedish Research CouncilSwedish Cancer SocietyEU, FP7, Seventh Framework Programme, 259796
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2017-12-05Bibliographically approved
Koos, B., Cane, G., Grannas, K., Löf, L., Arngården, L., Heldin, J., . . . Söderberg, O. (2015). Proximity-dependent initiation of hybridization chain reaction. Nature Communications, 6, Article ID 7294.
Open this publication in new window or tab >>Proximity-dependent initiation of hybridization chain reaction
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed) Published
Abstract [en]

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

National Category
Medical and Health Sciences Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568, 316929, 259796Swedish Foundation for Strategic Research Swedish Research Council
Available from: 2015-06-17 Created: 2015-06-17 Last updated: 2018-01-25Bibliographically approved
Mansouri, L., Sutton, L.-A., Ljungström, V., Bondza, S., Arngården, L., Bhoi, S., . . . Rosenquist, R. (2014). Recurrent Mutations within the Nfkbie gene: A Novel Mechanism for NF-kappa B Deregulation in Aggressive Chronic Lymphocytic Leukemia. Blood, 124(21)
Open this publication in new window or tab >>Recurrent Mutations within the Nfkbie gene: A Novel Mechanism for NF-kappa B Deregulation in Aggressive Chronic Lymphocytic Leukemia
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2014 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 124, no 21Article in journal, Meeting abstract (Other academic) Published
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-249073 (URN)000349242707052 ()
Available from: 2015-04-17 Created: 2015-04-10 Last updated: 2017-12-04Bibliographically approved
Clausson, C.-M., Söderberg, O., Arngården, L., Ishaq, O., Wählby, C., Nilsson, M. & Krzywkowski, T.Compaction of rolling circle amplification products increases signal strength and integrity.
Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal strength and integrity
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-217748 (URN)
Available from: 2014-02-04 Created: 2014-02-04 Last updated: 2018-06-08Bibliographically approved
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