Journal: International Journal of Molecular Sciences

Article Title: Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells

doi: 10.3390/ijms20215446

Figure Lengend Snippet: ( A ) High-temperature-required protein A2 (HtrA2) is found after staurosporine induction of apoptosis in the supernatant of human umbilical vein endothelial cells (HUVECs). ( B ) HtrA2 is significantly increased in the supernatant of HUVECs after H 2 O 2 -induced necrosis. Data is presented as dot plots with means and Standard Deviation(S.D). Significantly different vs. corresponding control with **** p < 0.0001, *** p <0.001 and ** p < 0.01.

Article Snippet: 17–19 h later, the cells were washed and incubated in medium with supplementary inductors; for experiments analyzing the extracellular release of HtrA2, 4 mM H 2 O 2 (Merck, Darmstadt, Germany) and 200 nM staurosporine (Abcam, Frankfurt, Germany) were used for 24 h. For experiments analyzing the extracellular effect of HtrA2, 2 μg/mL human recombinant HtrA2 (R&D Systems, Wiesbaden, Germany) +/− 2 μg/mL anti - HtrA2 antibody (R&D systems, Germany) was added to the medium.

Techniques: Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells

doi: 10.3390/ijms20215446

Figure Lengend Snippet: Extracellular HtrA2-induced apoptosis ( A ) but not necrosis ( B ) in HUVECs. HUVEC were stimulated with (2 µg/mL) of recombinant HtrA2, staurosporine (200 nM) or vehicle for a 24-h period. Results are presented as means with S.D. Significantly different compared to corresponding control with **** p < 0.0001 *** p < 0.001, ** p < 0.01 and * p < 0.05.

Article Snippet: 17–19 h later, the cells were washed and incubated in medium with supplementary inductors; for experiments analyzing the extracellular release of HtrA2, 4 mM H 2 O 2 (Merck, Darmstadt, Germany) and 200 nM staurosporine (Abcam, Frankfurt, Germany) were used for 24 h. For experiments analyzing the extracellular effect of HtrA2, 2 μg/mL human recombinant HtrA2 (R&D Systems, Wiesbaden, Germany) +/− 2 μg/mL anti - HtrA2 antibody (R&D systems, Germany) was added to the medium.

Techniques: Recombinant, Control

Journal: International Journal of Molecular Sciences

Article Title: Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells

doi: 10.3390/ijms20215446

Figure Lengend Snippet: Morphology of HUVEC treated with HtrA2, staurosporine, H 2 O 2 or untreated control. Morphology is visualised under microscope (magnification, 40×).

Article Snippet: 17–19 h later, the cells were washed and incubated in medium with supplementary inductors; for experiments analyzing the extracellular release of HtrA2, 4 mM H 2 O 2 (Merck, Darmstadt, Germany) and 200 nM staurosporine (Abcam, Frankfurt, Germany) were used for 24 h. For experiments analyzing the extracellular effect of HtrA2, 2 μg/mL human recombinant HtrA2 (R&D Systems, Wiesbaden, Germany) +/− 2 μg/mL anti - HtrA2 antibody (R&D systems, Germany) was added to the medium.

Techniques: Control, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells

doi: 10.3390/ijms20215446

Figure Lengend Snippet: Anti-HtrA2 antibodies partially inhibited extracellular HtrA2 and staurosporine-induced apoptosis. HUVEC cultures were incubated in n the presence and absence of staurosporine (200 nM), HtrA2 (2 μg/mL) and anti-HtrA2 (2 μg/mL). The percentage of apoptotic HUVECs was determined after 2 h by Annexin V staining. Results are presented as means with S.D that are significantly different to corresponding control with ** p <0.01 and * p < 0.05.

Article Snippet: 17–19 h later, the cells were washed and incubated in medium with supplementary inductors; for experiments analyzing the extracellular release of HtrA2, 4 mM H 2 O 2 (Merck, Darmstadt, Germany) and 200 nM staurosporine (Abcam, Frankfurt, Germany) were used for 24 h. For experiments analyzing the extracellular effect of HtrA2, 2 μg/mL human recombinant HtrA2 (R&D Systems, Wiesbaden, Germany) +/− 2 μg/mL anti - HtrA2 antibody (R&D systems, Germany) was added to the medium.

Techniques: Incubation, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells

doi: 10.3390/ijms20215446

Figure Lengend Snippet: Extracellular HtrA2 did not activate ICAM and VCAM. HUVEC cultures were incubated in HUVEC medium in the presence and absence of TNFα (10 ng), HtrA2 (2 μg/mL) and HtrA2+ TNFα. The expression of VCAM ( A ) and ICAM ( B ) in HUVECs was determined after 4-, 8- and 24h h by measuring the mean fluorescence intensity (MFI). Results are means with S.D. Significantly different with **** p < 0.0001 and * p < 0.05.

Article Snippet: 17–19 h later, the cells were washed and incubated in medium with supplementary inductors; for experiments analyzing the extracellular release of HtrA2, 4 mM H 2 O 2 (Merck, Darmstadt, Germany) and 200 nM staurosporine (Abcam, Frankfurt, Germany) were used for 24 h. For experiments analyzing the extracellular effect of HtrA2, 2 μg/mL human recombinant HtrA2 (R&D Systems, Wiesbaden, Germany) +/− 2 μg/mL anti - HtrA2 antibody (R&D systems, Germany) was added to the medium.

Techniques: Incubation, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells

doi: 10.3390/ijms20215446

Figure Lengend Snippet: Potential mechanism of HtrA2-mediated release and extracellular induction of apoptosis. This figure further describes already known mechanisms of intracellular induction of apoptosis by HtrA2. The intrinsic apoptotic pathway leads to the release of cytochrome c and HtrA2 from the intermembrane space of mitochondria into the cytosol. Cytochrome c induces apoptosis via caspase activation. HtrA2 proteolyses the inhibitory protein XIAP activating caspase 3/7 and 9 and subsequently induces apoptosis. In our study, apoptosis induction increased extracellular HtrA2 significantly. Extracellular HtrA2 was able to induce apoptosis in HUVEC. A possible mechanism might be the activation of the extrinsic pathway or through unknown mechanism; x-linked inhibitor of apoptosis protein (XIAP). brown arrows = possible mechanism of HtrA2 mediated apoptosis induction, brown dotted arrow = extracellular release of HtrA2, black arrows = activation, black T arrows = inhibition.

Article Snippet: 17–19 h later, the cells were washed and incubated in medium with supplementary inductors; for experiments analyzing the extracellular release of HtrA2, 4 mM H 2 O 2 (Merck, Darmstadt, Germany) and 200 nM staurosporine (Abcam, Frankfurt, Germany) were used for 24 h. For experiments analyzing the extracellular effect of HtrA2, 2 μg/mL human recombinant HtrA2 (R&D Systems, Wiesbaden, Germany) +/− 2 μg/mL anti - HtrA2 antibody (R&D systems, Germany) was added to the medium.

Techniques: Activation Assay, Inhibition

Journal: ESC Heart Failure

Article Title: Effects of elamipretide on skeletal muscle in dogs with experimentally induced heart failure

doi: 10.1002/ehf2.12408

Figure Lengend Snippet: Skeletal muscle fibre‐type distribution and inducible nitric oxide synthase isoform level in normal, HF‐CON ( n = 7), and HF + ELAM ( n = 7) dogs

Article Snippet: Primary antibodies for HtrA2 and cytochrome c were obtained from Santa Cruz, CA, antibody for porin from Abcam, Cambridge, MA, and antibodies for iNOS from LifeSpan Biosciences, Inc., Seattle, WA.

Techniques:

Journal: PLoS ONE

Article Title: HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

doi: 10.1371/journal.pone.0011380

Figure Lengend Snippet: Proteins uniquely associated with ARC-purified hmCD40 as determined by mass spectrometry analysis.

Article Snippet: Antibodies to HTRA2, cIAP1, SMAC/DIABLO, and actin were from Epitomics, Proteintech Group, BD Biosciences, and Chemicon International, respectively.

Techniques: Mass Spectrometry

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Increased expression and release of HtrA2 and p-HtrA2 from the mitochondria upon loss of Dusp26. A IHC staining of SNpc region showing expression of pS142-HtrA2 (left panel) or HtrA2 (right panel). Quantification of the number of p-HtrA2-positve cells per 1000 cells is presented. n = 5 mice/group. Scale bars p-HtrA2, 10 µm, HtrA2, 20 µm. B IHC staining of SNpc region showing colocalization of HtrA2 with TH in dusp26−/− mice. Quantification of the data is presented. n = 5 mice/group. Scale bars 20 µm. C Immunoblotting analyses of MB cytoplasmic and mitochondrial fractions showing the increased expression of HtrA2 in the cytoplasmic fraction isolated from the mutant mice. The expression of Hsp60 and Hsc70 indicate organelle purity. Quantification of the HtrA2 in the cytoplasmic fraction relative to WT is indicated in the right panel. n = 4–5 mice/group. D Immunoblotting analyses to detect Xiap expression in the MB and cortex of WT and dusp26−/− mice. β-actin is loading control. Quantification of the data relative to WT are presented in the right panel. n = 4–5 mice/group. In all relevant panels, bars are mean ± SEM. p values are indicated in each panel

Article Snippet: The following antibodies were used for IHC staining: Abcam: Dusp26 (b96429); Dako: GFAP (Z0334); Millipore: NeuN (MAB377), TH (AB152); Wako; Iba1 (019–19741); Sigma: TH (T2928); Santa Cruz Biotech: p-p38 (sc-58480), ERK1 (sc-94); Bioss: 8-OHDG (bs1278R); Assay Biotech: HtrA2 (B8515); CUSABIO: pS142-HtrA2 (CSB-PA008883).

Techniques: Expressing, Immunohistochemistry, Western Blot, Isolation, Mutagenesis, Control