Journal: Cell Communication and Signaling : CCS

Article Title: Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation

doi: 10.1186/s12964-025-02530-3

Figure Lengend Snippet: ADAM15 knockdown affects necroptosis but not apoptosis, NFκB, and MAPK signaling in U937 cells. a CRISPR Cas-9 mediated knockout of ADAM15 was monitored by WB using two different antibodies (R&D: MAB935 and HPA: HPA011633). Knockout (ko) cells are further referred to as ΔADAM15. Actin served as a loading control. n = 3 experiments performed. b ADAM15 knockout was validated by flow cytometry. The green curve represents staining using an anti-ADAM15 antibody (upper panel) or an isotype control (lower panel) in U937 wt cells. The red curve represents staining in U937 ΔADAM15 cells. The dashed non-coloured curves represent unstained cells. Right-shifted curves indicate higher fluorescence intensity. Representative data of n = 3 experiments are shown. c The degradation of IκB was monitored by WB. Band densities were quantified, and IkB:actin ratios are shown (green: U937 wt; red: U937 ΔADAM15). Representing n = 3 independent experiments. d The activation/phosphorylation of MAPK was monitored by WB. Band densities were quantified, and MAPK:actin ratios are shown (green: U937 wt; red: U937 ΔADAM15). Representative data of n = 3 experiments are shown. e Representative scatter plots of flow cytometry-based cell death analyses. The x-axis shows changes in fluorescence intensity due to increased Annexin V-FITC binding. The y-axis indicates increased fluorescence of 7AAD due to binding to DNA. Multiple ( n = 9) assays were quantified (right panel; green bars: WT; red bars: ΔADAM15). *: p = 0,05; ** p = 0,01; *** p = 0,001. f WB was used to monitor TC-mediated apoptosis (PARP-1 and caspases-3 and −8) and TCz-mediated necroptosis (MLKL oligomer). n = 3 experiments performed. Actin serves as a loading control

Article Snippet: Complexes were delivered to the nuclei of 5 × 10 3 cells using the nucleofection kits (U937: Kit C; Jurkat: Kit V) following the manufacturer’s instructions (Amaxa).

Techniques: Knockdown, CRISPR, Knock-Out, Control, Flow Cytometry, Staining, Fluorescence, Activation Assay, Phospho-proteomics, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation

doi: 10.1186/s12964-025-02530-3

Figure Lengend Snippet: ADAM15 knockdown affects necroptosis but not apoptosis induction in U937 cells mediated by TRAIL, FasL, TL1a, and Obatoclax. a-c Representative scatter plots of flow cytometry-based cell death analyses upon incubation with TRAIL (a), FasL (b), or TL1a ( c ) The x-axis shows changes in fluorescence intensity due to increased Annexin V-FITC binding. The y-axis indicates increased fluorescence of 7AAD due to binding to DNA. Multiple ( n = 3 per ligand) assays were quantified (right panels; green bars: WT; red bars: ΔADAM15) d Obatoclax mediated apoptosis (Oba) and necroptosis (Obaz) compared to TNF/BV-6 (TB: apoptosis) and TNF/BV-6/zVAD (TBz: necroptosis) were monitored by WB in U937 wt (left panel) and U937 ΔADAM15 cells (right panel). Represents n = 3 independent experiments. Tubulin serves as a loading control

Article Snippet: Complexes were delivered to the nuclei of 5 × 10 3 cells using the nucleofection kits (U937: Kit C; Jurkat: Kit V) following the manufacturer’s instructions (Amaxa).

Techniques: Knockdown, Flow Cytometry, Incubation, Fluorescence, Binding Assay, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation

doi: 10.1186/s12964-025-02530-3

Figure Lengend Snippet: Shotgun proteome analysis reveals regulated proteins upon ADAM15 knockout in U937 wt and U937 ΔADAM15 cells. a Volcano plot (p-value: 0.01; fdr: 0.01; fold-change cutoff 1: 0.3; fold-change cutoff 2: −0.3; min. samples for significance 0.75) shows proteins enriched in wt cells (red) versus U937 ΔADAM15 cells. The top 25 modulated proteins are indicated along with the selected proteins. The top annotated GSEA groups are shown: b ‘Regulation of endocytosis’, c ‘Regulation of autophagy’, d ‘Macroautophagy’, e ‘Organelle fusion’, f ‘Vacuole organization’, g ‘Regulation of mitochondrion organization’, h ‘Cell redox homeostasis’, i ‘Epigenetic regulation of gene expression’, and j ‘Mitotic cell cycle phase transition’. The upper panel shows the respective GO-termini, encircled by the involved proteins (Fold change is color-coded). The lower panels show the respective proteins as STRING-DB (V12.0) based networks. ADAM15 is highlighted for each network (dashed red circle)

Article Snippet: Complexes were delivered to the nuclei of 5 × 10 3 cells using the nucleofection kits (U937: Kit C; Jurkat: Kit V) following the manufacturer’s instructions (Amaxa).

Techniques: Knock-Out, Gene Expression, Sublimation

Journal: Cell Communication and Signaling : CCS

Article Title: Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation

doi: 10.1186/s12964-025-02530-3

Figure Lengend Snippet: Analysis of differential protein expression by WB and caspase-8 activity. a Depicts the validation of up- and down-modulation of selected proteins from the proteome analysis. b Representative (of n = 3) WB for RIPK1 probed with two different antibodies (cell signaling: #3493S and BD: 610,459). c Representative (of n = 3) WB analysis of various cell death-associated and non-related proteins. d Caspase-8 appears partially active in U937 ΔADAM15 compared to wt cells, indicated by reduced p55/44 and increased p43/41 and p18 (blue box – enhanced contrast). e Validation of increased basal Caspase-8 activity by flow cytometry. Dashed colorless curves indicate no staining. The green curve indicates wt cells stained with IETD-FITC, and the red curve indicates U937 ADAM15 cells with IETD-FITC. X-axis shift to the right side indicates increased fluorescence due to binding of IETD to active caspase-8 (p-18). Representative of n = 3 experiments. f Flow cytometry-based analysis of death receptor and ADAM15 surface expression. The non-coloured curves represent unstained and Strep-AF488-stained cells, the green curve WT, and the red curve ADAM15 cells. Representative data of n = 3 experiments

Article Snippet: Complexes were delivered to the nuclei of 5 × 10 3 cells using the nucleofection kits (U937: Kit C; Jurkat: Kit V) following the manufacturer’s instructions (Amaxa).

Techniques: Expressing, Activity Assay, Biomarker Discovery, Flow Cytometry, Staining, Fluorescence, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation

doi: 10.1186/s12964-025-02530-3

Figure Lengend Snippet: ADAM15 knockout in Jurkat cells results in abrogation of necroptosis induction and enhanced basal caspase-8 activity. a ADAM15 knockout was monitored by WB and b flow cytometry. The green curve represents staining using an anti-ADAM15 antibody (upper panel) or an isotype control (lower panel) in U937 wt cells. The red curve represents staining in Jurkat ΔADAM15 cells. The dashed non-coloured curves represent unstained cells. Right-shifted curves indicate higher fluorescence intensity. c degradation of IκB and d phosphorylation of MAPK are not affected by ADAM15 knockout. Band densities were quantified, and IκB:actin or MAPK:actin ratios are shown (green: Jurkat wt; red: Jurkat ΔADAM15). e WB analysis of cell death in Jurkat cells. f Representation of multiple ( N = 4) flow cytometry-based cell death analyses (green bars: WT; red bars: ΔADAM15). g WB analysis of Obatoclax (Oba) mediated cell death in Jurkat cells. TNF/BV-6 or TNF/BV-6/zVAD treatment served as control. h WB was probed for cell death-related and unrelated proteins, revealing reduced amounts of full-length RIPK1 (third panel, left) and pre-activated caspase-8 (first panel, right). i The enhanced basal activation of caspase-8 was validated using flow cytometry. The dashed colorless curves indicate no staining. The green curve indicates wt cells stained with IETD-FITC, and the red curve indicates U937 ADAM15 cells with IETD-FITC. X-axis shift to the right side indicates increased fluorescence due to binding of IETD to active caspase-8 (p-18). In a, c, d, e, and f, actin or tubulin served as loading controls. Where not stated otherwise, representative data of n = 3 experiments are shown

Article Snippet: Complexes were delivered to the nuclei of 5 × 10 3 cells using the nucleofection kits (U937: Kit C; Jurkat: Kit V) following the manufacturer’s instructions (Amaxa).

Techniques: Knock-Out, Activity Assay, Flow Cytometry, Staining, Control, Fluorescence, Phospho-proteomics, Activation Assay, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation

doi: 10.1186/s12964-025-02530-3

Figure Lengend Snippet: Exogenously expressed RIPK1 is degraded in U937 ΔADAM15 cells, and ADAM15 is partially located in lysosome-like organelles. a WB analysis of exogenously expressed RIPK1: accumulation of full-length RIPK1 in U937 wt cells, and moderately in U937 ΔADAM15 cells (black arrowhead). In U937 ΔADAM15 cells, RIPK1 is directly cleaved (grey arrowhead). Actin serves as a loading control. b Lamp-2, Cathepsin D, and also ADAM15 are enriched in the lysosome-containing fraction (LysIP), compared to total lysate or soluble proteins (SNT). nM represents the lysosome-depleted non-magnetic fraction remaining after the isolation procedure. p8 and p10 represent two different homogenization procedures. Nucleoporin p62 and actin serve as loading/purity controls. c Fluorescence microscopy monitoring ADAM15 (red) and Lamp-1 (green) reveals partial co-localization (arrowheads) of both proteins in U937 cells. Manders’ tM1/tM2 values are: 0.495/0.619 (upper left); 0.403/: 0.362 (upper right), (lower left) 0.434/0.377; (lower right) 0.302/0.371 (lower right). Values of tM1/tM2 > 0.5 are considered moderate, > 0.7 strong colocalized. DNA (blue) is stained with DAPI. Scale bar indicates 10 µm. Where not stated otherwise, representative data of n = 3 experiments are shown. d Working model of ADAM15-mediated control of necroptosis induction (Model created in BioRender)

Article Snippet: Complexes were delivered to the nuclei of 5 × 10 3 cells using the nucleofection kits (U937: Kit C; Jurkat: Kit V) following the manufacturer’s instructions (Amaxa).

Techniques: Control, Isolation, Homogenization, Fluorescence, Microscopy, Staining