Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 1 | Genome-wide screen identifies PACS-2 as an ADAM17 regulator. (a) Cellular model system used for the genome-wide siRNA screen. A total of 21,121 individual genes were knocked down using siRNAs and the effects on ADAM17-mediated shedding of AP-HB-EGF were measured by quantifying AP cell-surface staining after PMA stimulation. (b, c) siRNA-transfected AP-HB-EGF HT1080 (b) or AP-HB-EGF HeLa (c) cells were PMA-stimulated, and the cell medium was analysed for AP activity. The fold change in AP-HB-EGF release was calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value, and finally calculating the average of all individual experiments. Data in b were compiled from six individual experiments and in c from three individual experiments, each performed in triplicate. (d) AP-HB-EGF MCF-7 cells were siRNA- transfected and stimulated with PMA (left-hand panel) or ionomycin (right-hand panel). Conditioned medium was analysed for AP activity as in b and c. Data were compiled from four individual experiments, each performed in triplicate. In all cases, knockdown was confirmed by western blot. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance. **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Genome Wide, Staining, Transfection, Activity Assay, Negative Control, Knockdown, Western Blot

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 2 | PACS-2 regulates ADAM17-mediated shedding. (a) AP-HB-EGF was transiently expressed in control and Pacs2 / MEFs. Cells were PMA-stimulated, and the cell medium was analysed for AP activity. To overcome differences in transfection efficiency, the fraction of shed AP-HB-EGF was calculated as AP-HB-EGF released to the medium divided by the total amount of AP-HB-EGF in the medium and cell lysate. The fold change in AP-HB-EGF release was then calculated by setting the unstimulated negative control for each experiment to 1, normalizing the other raw data to this value and finally calculating the average of all individual experiments. The PMA-stimulated fold increase in AP-HB-EGF shedding was depicted for each cell line. Data were compiled from four individual experiments, each performed in triplicate. (b) AP-HB-EGF was expressed in Pacs2 / MEFs together with green fluorescent protein (GFP) or PACS-2-HA and shedding analysed as in a. Data were compiled from three individual experiments, each performed in triplicate. (c–e) siRNA-transfected MDA-MB-231 cells were stimulated with PMA (c,d) or TNF-a (e). The medium was analysed by ELISA for shed HB-EGF (c) or TGF-a (d,e), shown as concentrations in pg ml 1. In all cases, data were compiled from three individual experiments, each performed in triplicate. (f) Representative western blots showing knockdown in cells used for ELISA. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Control, Activity Assay, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 3 | PACS-2 regulates cell-surface availability of mature ADAM17. (a,b) MDA-MB-231 cells were siRNA-transfected and PMA-stimulated. Cells were surface biotinylated and analysed by western blot. ADAM17 levels were normalized to input actin and fold changes were calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value and finally calculating the average of all individual experiments. Cell-surface levels of mature ADAM17 (a) and total cellular levels of pro and mature ADAM17 (b) are shown with data compiled from four individual experiments. (c,d) Unstimulated cell-surface levels of mature ADAM17 (c) and total cellular levels of pro and mature ADAM17 (d) in control and Pacs2 / MEFs were analysed as in a and b. Data were compiled from four individual experiments in c and three individual experiments in d. (e) ADAM17 was immunoprecipitated from unstimulated control and Pacs2 / MEFs and its activity assessed using an ADAM17 quenched fluorescence peptide substrate. The graph shows the average gradient (fluorescence units/time) of the linear regressions describing enzymatic activity. The data were compiled from three individual experiments, each performed in triplicate. (f) Lysates from control and Pacs2 / MEFs were treated with EndoH or PNGase F and the effects on ADAM17 examined by western blot. The images shown are derived from the same blot but different exposures (indicated by separate panels). The blot shown is representative of three individual experiments. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Transfection, Western Blot, Negative Control, Control, Immunoprecipitation, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 4 | PACS-2 interacts with ADAM17 in endocytic compartments. (a,b) ADAM17 (a) or PACS-2 (b) was immunoprecipitated (IP) from unstimulated and PMA-stimulated MDA-MB-231 cells, and co-immunoprecipitation (co-IP) of PACS-2 (a) or ADAM17 (b) detected by western blot. The blots shown are representative of three individual experiments and # denotes a nonspecific band. (c) The effect of PMA stimulation on PACS-2/ADAM17 co-immunoprecipitation in a and b was quantified. The amount of co-immunoprecipitated PACS-2 (a) or mature ADAM17 (b) was normalized to the amount of immunoprecipitated mature ADAM17 (a) or PACS-2 (b). The unstimulated negative control for each experiment was then set to 1, the other raw data were normalized to this value and finally the average of all individual experiments was calculated. Data were analysed by unpaired two-tailed Student’s t-test. (d) MDA-MB-231 cells were transfected with siRNA and subjected to PLA with or without PMA stimulation. The experiment was performed 10 times in duplicate. Two experiments included the knockdown controls. Scale bar, 7 mm. (e) Before PLA, MDA-MB-231 cells were left unstimulated or treated with PMA and subsequently allowed to internalize fluorescent transferrin for 5 min. Scale bar, 14 mm. Fluorescence intensities were quantified along the lines on the enlarged images and depicted on the graphs. The experiment was performed four times in duplicate.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Two Tailed Test, Transfection, Knockdown, Fluorescence

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 5 | PACS-2 diverts endocytosed ADAM17 away from degradative pathways. (a) MDA-MB-231 cells were surface labelled using cleavable biotin. Labelled cell-surface proteins were allowed to internalize for 30 min and remaining biotinylated proteins on the cell surface were removed (Intern.). Internalized proteins were then allowed to recycle for 30 min, after which recycled protein was stripped from the cell surface (Recyc.). The amount of cell-surface ADAM17 was first normalized to input actin. The percentage internalization was calculated relative to total cell-surface ADAM17 levels (Total). Recycling was determined by first subtracting the amount of biotinylated protein left after recycling from the amount of internalized protein, and then calculated as a percentage of internalized protein. To verify proper stripping of cell-surface proteins, a dish kept at 4 C was processed in parallel (Strip). Data were compiled from five individual experiments. (b) MDA-MB-231 cells were surface labelled using non-cleavable biotin, lysed at time zero, or incubated for 4 h at 37 C to allow internalization and degradation of surface proteins. Percentage degradation was calculated by normalizing the amount of cell-surface ADAM17 to input actin, and dividing the amount remaining after 4 h with the amount present at time zero. Data were compiled from three individual experiments. The images shown are derived from the same blot and same exposure, but have been cropped for clarity (indicated by a separation line). On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by unpaired two-tailed Student’s t-test. *Po0.05.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Stripping Membranes, Incubation, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 8 | A model of PACS-2 regulation of ADAM17 and EGFR activity. The ADAM17 proform mainly resides in the secretory apparatus, and during its transit to the cell surface, furin-mediated cleavage generates the mature (active) form. We propose that PACS-2 controls ADAM17 cell-surface availability by diverting endocytosed ADAM17 away from degradative pathways and towards sustained ErbB ligand shedding and EGFR activation. PACS-2 is therefore an important regulator of auto, para and juxtacrine ErbB signalling in vivo.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Activity Assay, Activation Assay, In Vivo

Journal: BMC Gastroenterology

Article Title: Liver protective effect of ursodeoxycholic acid includes regulation of ADAM17 activity

doi: 10.1186/1471-230X-13-155

Figure Lengend Snippet: UDCA interferes with ADAM17 maturation. HepG2 cells were either left untreated (control) or pretreated with 200 μmol/l UDCA (UDCA) or with 10 nmol/l metalloproteinase inhibitor TAPI-2 (TAPI) for 2 hours. Cells were then either stimulated with 10 nmol/l PMA (PMA) for an additional 24 hours or left non-stimulated. (A) Cell lysates from treated and control cells were analyzed by immunoblotting with anti-ADAM17 antibodies. Equal amounts of proteins were loaded in each lane; representative Western blots from three independent experiments are shown. (B) Signal intensities of mature ADAM17 bands (85 kDa), which were densitometrically determined in three independent experiments (including the one shown), were normalized to immature ADAM17 form (130 kDa). Mean values ± SEM are shown (n = 3). *p < 0.05; **p < 0.01. (C) mRNA was isolated from HepG2 cells as described in the text. The relative expression levels of ADAM17 were followed by qRT-PCR. Expression was normalized to GAPDH and expressed as fold change of control sample. Mean values ± SEM are shown (n = 3). *p < 0.05.

Article Snippet: The human cDNA clone of full-length ADAM17 in the pCMV6-XL4 vector was obtained from OriGene (SC316426; OriGene Technologies, Rockville, MD).

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

Journal: Scientific Reports

Article Title: Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

doi: 10.1038/s41598-021-89063-0

Figure Lengend Snippet: Clinical features of patients molecularly diagnosed with NISBD1 in presented and reported cases.

Article Snippet: Although the full-length coding sequence of human ADAM17 could be amplified from RNA extracted from human cells through RT-PCR, it was impossible to clone PCR products into plasmids and stably replicate them in Escherichia coli due to the spontaneously and frequently occurring SNVs, InDels and/or rearrangements in ADAM17 , as warned by OriGene Technologies (Rockville, MD, USA; https://www.origene.com/catalog/cdna-clones/expression-plasmids/sc316426/adam17-nm_003183-human-untagged-clone ).

Techniques: Infection, Virus, Variant Assay

Journal: Scientific Reports

Article Title: Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

doi: 10.1038/s41598-021-89063-0

Figure Lengend Snippet: ( a ) Partial sequence chromatograms of exons 14 and 15 of a disintegrin and metalloprotease 17 ( ADAM17 ) in the genomic DNA of patients (II:1 and II:2) and their parents (I:1 and I:2). Nucleotide and corresponding amino acid sequences of WT and mutant ADAM17 are also shown. Numbers indicate transformed codons by single-base substitutions (red arrows). P proband. ( b ) Partial sequence chromatograms of exons 14 and 15 of ADAM17 in the mRNA of peripheral blood leucocytes from the proband (patient II:1). Both WT and variant alleles were detected at the same level, suggesting that ADAM17 was equally transcribed from both alleles. Numbers indicate transformed codons by single-base substitutions (red arrows). ( c ) Comparison of partial amino acid sequences of ADAM17 in human (NP_003174.3), chimp (XP_515293.2), rhesus monkey (XP_002799185.1), mouse (NP_033745.4), chicken (NP_001008682.1), Xenopus tropicalis (NP_001182159.1), fugu (XP_011616093.2), zebrafish (Adam17a, NP_955967.1), fruit fly (Tace, NP_733334.1) and Caenorhabditis elegans (adm-4, NP_509318.1). Conserved amino acids are highlighted. Mutated amino acids are shown by red arrowheads. Numbers indicate codons of mutated amino acids. Bar indicates the highly conserved thioredoxin CXXC motif. ( d ) Schematic representation of ADAM17. Domains are depicted approximately to the scale adapted from the human reference amino acid sequence (NP_003174.3). Red arrows show the position corresponding to variants detected in patients in a compound heterozygous state and both parents in a heterozygous state. CANDIS conserved ADAM17 dynamic interaction sequence, MPD membrane proximal domain, CT C-terminal cytoplasmic tail, D disintegrin-like domain, M metalloprotease domain, Pro pro-domain, S signal sequence, TM transmembrane domain. Blue bar indicates the cysteine-rich (Cys-rich) region. The ADAM17 precursor (left) is converted into a mature active form (right) through the enzymatic removal of the signal sequence and pro-domain. Activated ADAM17 is trafficked to the cell surface, where shedding of substrates can occur.

Article Snippet: Although the full-length coding sequence of human ADAM17 could be amplified from RNA extracted from human cells through RT-PCR, it was impossible to clone PCR products into plasmids and stably replicate them in Escherichia coli due to the spontaneously and frequently occurring SNVs, InDels and/or rearrangements in ADAM17 , as warned by OriGene Technologies (Rockville, MD, USA; https://www.origene.com/catalog/cdna-clones/expression-plasmids/sc316426/adam17-nm_003183-human-untagged-clone ).

Techniques: Sequencing, Mutagenesis, Transformation Assay, Variant Assay, Comparison, Membrane

Journal: Scientific Reports

Article Title: Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

doi: 10.1038/s41598-021-89063-0

Figure Lengend Snippet: ( a , b ) mRNA and protein expression of exogenously transfected a disintegrin and metalloprotease 17 ( ADAM17 ) in Adam10/17 double-knockout mouse embryonic fibroblasts ( Adam10 / 17 −/− mEFs). ( a ) Adam10/17 −/− mEFs were transfected with FLAG-syn-hADAM17-WT, FLAG-syn-hADAM17-C567R, or FLAG-syn-hADAM17-C600Y and incubated for 48 h. Amounts of exogenous ADAM17 mRNA were measured by qRT-PCR, using GAPDH mRNA as an endogenous control. Values are expressed as fold changes (mean ± SD, n = 3) compared with respective values in cells transfected with syn-hADAM17-WT. ( b ) Transfected cells were lysed in M-PER buffer supplemented with a protease inhibitor. Panels show representative results from three independent western blots analysing the expression of FLAG-tagged ADAM17 and β-actin. Full-length blots were presented in Supplementary Figure a ( c , d ) Phorbol 12-myristate 13-acetate (PMA)-stimulated shedding activity for various substrates in Adam10/17 − /− mEFs exogenously transfected with ADAM17. ( c ) Adam10/17 −/− mEFs were co-transfected with pFLAG-syn-hADAM17 expression constructs and AP-tagged TNF-α, TGF-α, or HB-EGF expression constructs. Relative AP activity released into the media of transfected Adam10/17 −/− mEFs during the 2-h stimulation with PMA (20 ng/mL) in the presence or absence of the metalloprotease inhibitor BB94 (1 μM). Data represent three independent experiments. Error bars indicate mean ± SD. * P < 0.05. ( d ) Adam10/17 −/− mEFs were co-transfected with pFLAG-syn-hADAM17 expression constructs and AP-tagged TNF-α or TGF-α expression constructs. Relative AP activity released into the media of transfected Adam10/17 −/− mEFs during the 2-h stimulation with PMA (20 ng/mL) in the presence or absence of the human ADAM17 inhibitory antibody (15 μg/mL). Data represent three independent experiments. Error bars indicate mean ± SD. * P < 0.05.

Article Snippet: Although the full-length coding sequence of human ADAM17 could be amplified from RNA extracted from human cells through RT-PCR, it was impossible to clone PCR products into plasmids and stably replicate them in Escherichia coli due to the spontaneously and frequently occurring SNVs, InDels and/or rearrangements in ADAM17 , as warned by OriGene Technologies (Rockville, MD, USA; https://www.origene.com/catalog/cdna-clones/expression-plasmids/sc316426/adam17-nm_003183-human-untagged-clone ).

Techniques: Expressing, Transfection, Double Knockout, Incubation, Quantitative RT-PCR, Control, Protease Inhibitor, Western Blot, Activity Assay, Construct

Journal: Scientific Reports

Article Title: Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

doi: 10.1038/s41598-021-89063-0

Figure Lengend Snippet: ( a ) Effect of variants on the phorbol 12-myristate 13-acetate (PMA)-induced maturation of a disintegrin and metalloprotease 17 (ADAM17). Adam10/17 double-knockout mouse embryonic fibroblasts ( Adam10/17 −/− mEFs) were transfected with pFLAG-syn-hADAM17 expression constructs, incubated for 48 h and treated with PMA (100 ng/mL) in serum-free medium for 5 min. After subsequent incubation at 37 °C in DMEM for indicated times, cells were lysed in RIPA buffer supplemented with a protease inhibitor. Panels show representative results from three independent western blots analysing the expression of FLAG-tagged mature ADAM17 (mADAM17) and its preform (pADAM17) and an internal control β-actin (ACTB). The ratio of the densitometric signal of mADAM17 and pADAM17 was shown at the botttom. Full-length blots were presented in Supplementary Figure b. ( b ) Effect of variants on PMA-induced cell-surface localisation of mature ADAM17. HEK293 cells transfected with pFLAG-syn-hADAM17 expression constructs and incubated for 48 h were treated with PMA (100 ng/mL) in serum-free medium for 5 min. After incubation at 37 °C in DMEM for indicated times, cell-surface proteins were biotinylated and harvested. Biotinylated proteins precipitated using streptavidin beads were immunoblotted. The panel shows the representative result from three independent experiments. A full-length blot with a size marker image was presented in Supplementary Figure c. ( c ) Quantification of HiBiT-tagged ADAM17 localised on the cell surface. Adam10 / 17 −/− mEFs transfected with pHiBiT-ADAM17-WT, pHiBiT-ADAM17-C567R, or pHiBiT-ADAM17-C600Y and incubated for 48 h in 96-well microplates were treated with PMA (20 ng/mL) for 2 h. Extracellular and lytic luciferase activities were measured as described in the “ ” section (Supplementary Figure B). Values are expressed as the ratio of extracellular or lytic luminescence intensity for each well (mean ± SD, n = 6). * P < 0.05. Data are representative of at least three experiments with similar results.

Article Snippet: Although the full-length coding sequence of human ADAM17 could be amplified from RNA extracted from human cells through RT-PCR, it was impossible to clone PCR products into plasmids and stably replicate them in Escherichia coli due to the spontaneously and frequently occurring SNVs, InDels and/or rearrangements in ADAM17 , as warned by OriGene Technologies (Rockville, MD, USA; https://www.origene.com/catalog/cdna-clones/expression-plasmids/sc316426/adam17-nm_003183-human-untagged-clone ).

Techniques: Double Knockout, Transfection, Expressing, Construct, Incubation, Protease Inhibitor, Western Blot, Control, Marker, Luciferase

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Depletion of cellular cholesterol and lipid rafts increases shedding of CD30.

doi: 10.4049/jimmunol.172.7.4324

Figure Lengend Snippet: FIGURE 6. Localization of CD30 in membrane rafts. A–C, Membrane rafts of 3 107 L428 cells were isolated by applying cell lysates to a sucrose gradient and immunoblotted under nonreducing conditions. The indicated molecular mass markers were internal sCD30 standard (90 kDa) for determination of CD30 and commercially available TACE ectodomain standard (75 kDa) for determination of TACE. A, Proteins of fractions 3–11 were separated by nonreducing SDS-PAGE and immunoblotted with a mixture of HRP-labeled anti-CD30 Ki-2 and Ki-4 mAb. B, Proteins of fractions 3–5 (raft fractions) and 9–11 (nonraft fractions) were separated by nonreducing SDS-PAGE and immunoblotted with HRP-labeled anti- TACE MAB9302 mAb. As a control, the TACE-ectodomain standard was used. C, Two microliters of each fraction of L428 cells were dot blotted and stained with peroxidase-labeled cholera toxin subunit B as a positive control for lipid rafts. In fractions 3–5, rafts were isolated. D, L428 cells (1.5 107 per milliliter) were incubated in prewarmed RPMI 1640 con- taining 0.1% BSA in the absence or presence of MCD (10 mM) or PMA (5 ng/ml) at 37°C for 5 or 60 min as indicated. Membrane rafts of 3 107

Article Snippet: The anti-TACE mAb MAB9302, the recombinant human TACE ectodomain Western blotting standard, and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-3 were purchased from R&D Systems (WiesbadenNordenstadt, Germany).

Techniques: Membrane, Isolation, SDS Page, Labeling, Control, Staining, Positive Control, Incubation

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 1 | Shear stress induces most prominently iRhom1. HUVECs were cultured for 24 h under different flow conditions resulting in the indicated levels of laminar shear stress. Cells were then analyzed for mRNA expression of KLF2 (A), NOS3 (B), RHBDF1 (C), RHBDF2 (D), ADAM17 (E), and ADAM10 (F) in relation to a reference gene index consisting of GAPDH and TBP. The three independent experiments were performed with HUVECs from three different donors. A static control (0 dyn/cm2) was made for each shear stress level. All static controls were merged in one column for clarity. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the static control are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Shear, Cell Culture, Expressing, Control, Standard Deviation

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 2 | High physiological shear stress induces most prominently iRhom1 in endothelial cells from different vascular beds. HUVECs, HUAECs, HAMECs, and HPMECs were cultured for 24 h with a laminar shear stress of 30 dyn/cm2. Cells were then analyzed for mRNA expression of KLF2 (A), NOS3 (B), RHBDF1 (C), RHBDF2 (D), ADAM17 (E), and ADAM10 (F) in relation to a reference gene index consisting of GAPDH and TBP. The three independent experiments were performed with HUVECs and HUAECs from three different donors and HAMECs and HPMECs from two different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the static control are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001). Significant differences between the different endothelial cell types were marked with different letters (a, b, c, and d). Each group with one letter is significantly different from a group with another letter.

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Shear, Cell Culture, Expressing, Standard Deviation, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 3 | Shear stress-mediated iRhom1 mRNA expression is partially suppressed by MEK5 inhibition. HUVECs were pretreated with DMSO or 10 µM of the MEK5 inhibitor BIX02189 for 2.5 h and cultured for 24 h with a laminar shear stress of 30 dyn/cm2 in the presence of DMSO or BIX02189. Cells were then analyzed for mRNA expression of KLF2 (A), NOS3 (B), RHBDF1 (C), RHBDF2 (D), ADAM17 (E), and ADAM10 (F) in relation to a reference gene index consisting of GAPDH and TBP. At least four independent experiments were performed with HUVECs from four different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the corresponding static control are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001) and significant differences between DMSO and the MEK5 inhibitor are indicated as hashes (#p < 0.05, ##p < 0.01, and ###p < 0.001).

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Shear, Expressing, Inhibition, Cell Culture, Standard Deviation, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 5 | Inflammatory cytokines exclusively induce the mRNA expression of iRhom2 in endothelial cells. HUVECs were treated for 24 h with the indicated concentrations of TNFα, IFNγ or the combination of TNFα and IFNγ. Cells were then analyzed for mRNA expression of RHBDF1 (A), RHBDF2 (B), and ADAM17 (C). Three independent experiments were performed with HUVECs from three different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the untreated control cells are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001) and significant differences between TNFα and IFNγ treated cells to the corresponding cells treated with TNFα alone are indicated as hashes (#p < 0.05, ##p < 0.01, and ###p < 0.001).

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Standard Deviation, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 6 | TNFα-induced iRhom2 expression is mainly controlled by AP-1 in endothelial cells. HUVECs were pretreated with DMSO, 10 µM of the AP-1 inhibitor SR11302 (A–D) or 3 µM of the NFκB inhibitor Bay11-7082 (E–H) for 1 h and cultured for 24 h with 10 ng/ml TNFα, IFNγ or the combination of TNFα and IFNγ in the presence of DMSO, SR11302, or Bay11-7082. Cells were then analyzed for mRNA expression of RHBDF1 (A,E), RHBDF2 (B,F), ADAM17 (C,G), and ADAM10 (D,H). Five independent experiments were performed with HUVECs from five different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the corresponding untreated control cells are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001) and significant differences between DMSO and SR11302 or Bay11-7082 are indicated as hashes (#p < 0.05, ##p < 0.01, and ###p < 0.001).

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Cell Culture, Standard Deviation, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 7 | Shear stress and TNFα induce iRhom1 or iRhom2 in an independent manner. HUVECs were cultured for 24 h under static conditions or with a shear stress of 30 dyn/cm2 and subsequently stimulated with or without 10 ng/ml TNFα for another 24 h with or without flow. Cells were then analyzed for mRNA expression of RHBDF1 (A), RHBDF2 (B), ADAM17 (C), and ADAM10 (D). Four independent experiments were performed with HUVECs from four different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the corresponding static control are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001) and significant differences between untreated control cells and cells treated with TNFα are indicated as hashes (#p < 0.05, ##p < 0.01, and ###p < 0.001).

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Shear, Cell Culture, Expressing, Standard Deviation, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 8 | Shear stress- and TNFα-mediated induction of iRhom1 and iRhom2 lead to increased active ADAM17 on the cell surface. HUVECs were cultured for 24 h under static conditions or with a shear stress of 30 dyn/cm2 and subsequently stimulated with or without 10 ng/ml TNFα for another 24 h with or without shear stress. Cells were then analyzed for ADAM17 protein expression (A–C) and ADAM17 surface expression (D,E). The concentrated supernatant was analyzed for levels of soluble JAM-A (F). (A–C) Western blot results are shown as the ratio of the densitometric signal of total ADAM17 (tADAM17) and GAPDH (A), as the ratio of the densitometric signal of mature ADAM17 (mADAM17) and pro ADAM17 (pADAM17) (B), and as representative blot (C). (D,E) Results of the flow cytometric analysis are shown as geometric mean of the fluorescence intensity representing the relative ADAM17 surface expression (D) and as representative histogram (E). (F) Results of the JAM-A ELISA are presented as concentration of soluble JAM-A in pg/ml. Four independent experiments were performed with HUVECs from four different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the corresponding static control are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001) and significant differences between untreated control cells and cells treated with TNFα are indicated as hashes (#p < 0.05, ##p < 0.01, and ###p < 0.001).

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Shear, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: FIGURE 9 | Proposed model for the regulation of iRhom1 and iRhom2 in endothelial by shear stress and TNFα and the consequences for ADAM17 activity on the cell surface. While shear stress mainly induces iRhom1 mRNA expression, which is partially controlled via MEK5 and KLF2, TNFα exclusively induces iRhom2 mRNA expression in endothelial cells predominantly via AP-1. Both iRhoms contribute to increased ADAM17 maturation and surface expression or may even stabilize mature ADAM17 on the cell surface. This can then lead to increased shedding of ADAM17 substrates, such as JAM-A depending on the stimulatory condition.

Article Snippet: Mouse monoclonal antibody against the mature form of human ADAM17 (clone 111633) and mouse IgG1 isotype control were from R&D Systems (Wiesbaden, Germany).

Techniques: Shear, Activity Assay, Expressing