Increasing knowledge about the heterogeneity of mRNA expression within cell populations highlights the need to study transcripts at the level of single cells. We present a method for detection and ...genotyping of individual transcripts based on padlock probes and in situ target-primed rolling-circle amplification. We detect a somatic point mutation, differentiate between members of a gene family and perform multiplex detection of transcripts in human and mouse cells and tissue.
Hybridization Chain Reaction (HCR) is a technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on every ...hairpin being metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which puts a strong demand on oligonucleotide quality. We show how further purification can greatly increase polymerization potential. It was found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than a non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.
•Hybridization Chain Reaction (HCR) is highly sensitive to oligonucleotide quality.•Polymerization potential of HCR varies over production batches.•With purification, polymerization in HCR can be improved.•Purification increases stain strength several fold in in situ applications.
The discovery of oestrogen receptor β (ERβ/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERα (ESR1) raised hopes for improved ...endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERβ antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ERβ in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERβ protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.
Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues ...to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.
The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor ...(EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.
•EGFR phosphorylation and dimerization increases after cholesterol removal.•EGF-dependent activation of downstream AKT and ERK1/2 is reduced upon cholesterol removal.•EGFR and PDGFR-β may internalize separately but later coalesce in perinuclear vesicles.•EGFR and PDGFR-β deviate in their clathrin-independent internalization.
It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor ...tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor β (PDGFR-β) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-β-cyclodextrin (MβCD) did not affect PDGFR-β activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MβCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.
•PDGFR-β activation is affected by cholesterol depletion in a cell specific manner.•Cholesterol depletion impairs AKT and SRC signaling downstream of PDGFR-β in U2OS.•Cholesterol depletion abolish ERK signaling downstream of PDGFR-β in BJ-hTERT.•Cholesterol depletion affect cell morphology differently depending on cell type.
The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) ...has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.
•We used gene silencing to study the roles of protein disulphide-isomerase A3 (PDIA3).•PDIA3 has been proposed to play a role in vitamin D-mediated signalling.•PDIA3 silencing affected growth in prostate cell lines independently of vitamin D.•PDIA3 silencing affected VDR target genes, including CYP24A1.•Thermostability of PDIA3 was increased by presence of active vitamin D.
•Introducing a new protocol Flash-comet assay, with improved sensitivity and speed.•Low conductivity solution based on LiOH instead of NaOH-based solution.•Changing from lysis at pH 10 to 8.5 lowers ...the overall level of DNA-damage.
Evaluation of primary DNA-damage is one way to identify potential genotoxic agents and for this purpose the Comet assay has, for the last decades, been used to monitor DNA single strand and double strand breaks in individual cells. Various attempts have been made to modify the different steps in the in vitro protocol for the Comet assay in order to improve its sensitivity. However, to the best of our knowledge, nobody has tried to replace the traditionally used NaOH-based electrophoresis solution (pH > 13), with another type of solution. In the present paper, using TK-6 cells exposed to different concentrations of H2O2 or ionizing radiation, we present evidence clearly showing that a low-conductive LiOH-based electrophoresis solution at pH 12.5, and a more gentle lysis procedure, significantly improved both the speed and sensitivity of the assay. The new approach, which we call the Flash-comet, is based on a lysis buffer at pH 8.5, an unwinding time of 2.5 min in a LiOH solution without EDTA at pH 12.5, and an electrophoresis time of 1 min at 150 V (5 V/cm) using the same solution.
Abstract
In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks ...(SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks.
Graphical Abstract
Graphical Abstract
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 TGFβ. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFβ (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 TGFβ stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFβ 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 TGFβ signaling pathways.
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•Protein–protein interactions studied with single cell and subcellular resolution using in situ proximity ligation.•Density-dependent formation of YAP/TAZ–Smad2/3 complexes in response to TGFβ stimulation.•No YAP/TAZ–Smad2/3 complexes were observed in HT29 cells lacking Smad4.
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