Journal: bioRxiv

Article Title: Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

doi: 10.1101/290718

Figure Lengend Snippet: ( a ) Cell viability of KMB7 wildtype and KBM7 FADD - cells treated for 24h with 100 ng/ml TNFα, 1 µM SMAC mimetic birinapant, 50 µM Nec-1s and 10 µM z-VAD-FMK as indicated. ( b ) Cell viability of KBM7 FADD - cells treated for 24h with 100 ng/ml TNFα, 10 µM SMAC mimetic or 100 ng/ml TNFα and 1 µM SMAC mimetic, and the indicated inhibitors. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo) and normalized to untreated control. Data represent mean value ± s.d. of two independent experiments performed in triplicates. ( c ) Schematic overview summarizing the genetic screens presented in this study.

Article Snippet: Interestingly, we found that treatment with the SMAC mimetic birinapant alone sufficed to induce necroptosis in KBM7 FADD - cells ( ).

Techniques:

Journal: bioRxiv

Article Title: Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

doi: 10.1101/290718

Figure Lengend Snippet: ( a-c ), Circos plots of haploid genetic screens in KBM7 FADD - cells with necroptosis induction by 10 µM SMAC mimetic birinapant ( a ), 100 ng/ml TNFα ( b ), and 1 µM SMAC mimetic and 100 ng/ml TNFα combined ( c ). Each dot represents a mutagenized gene identified in the resistant cell population, dot size corresponds to the number of independent insertions identified for each gene and distance from center indicates the significance of enrichment compared to an unselected control data set. Hits with an adjusted p -value <10 -10 are labelled. ( d ) Bubble plot depicting the top hits over all three screens ranked according to adjusted p -value. Bubble size corresponds to the number of independent insertions identified and colour gradient reflects the significance of enrichment. ( e-f ), Multi-colour competition assay (MCA) of KBM7 FADD - SpCas9 cells transduced with a GFP marker (GFP + ) and sgRNAs targeting either SLC39A7 or RIPK1 ( e ), SP1 or TNFR2 ( f ), or Renilla luciferase ( sgRen ) as control, against cells transduced with sg Ren and an mCherry marker (mCherry + ). The cell populations were mixed at 1:1 ratio, treated with SMAC mimetic (1 µM) or TNFα (10 ng/ml), and analyzed after 14 days by flow cytometry. Data represent mean value ± s.d. of two independent experiments performed in duplicates, n.d. (not determined) indicates wells with no outgrowth.

Article Snippet: Interestingly, we found that treatment with the SMAC mimetic birinapant alone sufficed to induce necroptosis in KBM7 FADD - cells ( ).

Techniques: Competitive Binding Assay, Transduction, Marker, Luciferase, Flow Cytometry

Journal: bioRxiv

Article Title: Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

doi: 10.1101/290718

Figure Lengend Snippet: ( a - b ) Circos plots of genome-scale SAM screens in KBM7 FADD - SAM cells with necroptosis induction by TNFα ( a ) or SMAC mimetic birinapant ( b ). For each stimulus, screens were performed at low and high concentrations (TNFα: 10 ng/ml (green) or 100 ng/ml (purple); birinapant: 0.1 µM (blue) or 1 µM (orange)). Screen analysis was performed by identifying differentially enriched sgRNAs using DESeq2 and then aggregating sgRNAs to genes using Gene Set Enrichment Analysis. Identified hits are ranked according to the adjusted p -value of enrichment (–log10(padj). Bubble size corresponds to the average log2 fold-change (aLFC) of enrichment, color indicates the number of significantly enriched sgRNAs. Screens were performed in duplicate except the high concentration of Birinipant in simplicate. (c-d) Cell viability in KBM7 FADD - SAM cells transduced with sgRNA targeting TNIP1, BIRC3 or Renilla luciferase (Ren). Cells were treated as indicated for 72h and viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. ( e ) KBM7 FADD - CRISPR-SAM cells transduced with the specified sgRNAs were lysed and subjected to immunoblotting with the indicated antibodies.

Article Snippet: Interestingly, we found that treatment with the SMAC mimetic birinapant alone sufficed to induce necroptosis in KBM7 FADD - cells ( ).

Techniques: Concentration Assay, Transduction, Luciferase, CRISPR, Western Blot

Journal: RSC Chemical Biology

Article Title: The chemical biology of IL-12 production via the non-canonical NFkB pathway

doi: 10.1039/d0cb00022a

Figure Lengend Snippet: (a) Schematic of IL-12 modulation of checkpoint therapy through T-cell:dendritic cell cross talk. aPD-1 treatment induces IFNγ in T-cells, which promotes production of IL-12 in dendritic cells. IL-12 then further activates anti-tumor T-cells. IL-12 activates other cell types as well, which leads to additional anti. (b) Schematic of the non-canonical NFkB pathway. Activation of CD40, either through CD40L binding or through IFNγ signaling, disrupts the TRAF2-TRAF3-cIAP complex, allowing NIK levels to rise. NIK then promotes p100 processing via IKKa, which leads to an active RelB-p52 complex that can translocate to the nucleus and cause transcription of IL-12. (c) Description of various signaling nodes in the non-canonical NFkB pathway.

Article Snippet: Most cIAP inhibitors are mimetics of second mitochondria-derived activator of caspases (SMAC).

Techniques: Activation Assay, Binding Assay

Journal: RSC Chemical Biology

Article Title: The chemical biology of IL-12 production via the non-canonical NFkB pathway

doi: 10.1039/d0cb00022a

Figure Lengend Snippet: (a) Structure of various cIAP inhibitors (left), CD40 agonist (middle), and rimiducid (right), a tool compound used in cellular therapy. (b) Table of various clinical trials highlighting agents that modulate non-canonical NFkB signaling.

Article Snippet: Most cIAP inhibitors are mimetics of second mitochondria-derived activator of caspases (SMAC).

Techniques: Clinical Proteomics

Journal: RSC Chemical Biology

Article Title: The chemical biology of IL-12 production via the non-canonical NFkB pathway

doi: 10.1039/d0cb00022a

Figure Lengend Snippet: Sample screen for IL-12 inducers in murine dendritic cells. Several cIAP inhibitors scored high in this assay. Reproduced from Koch et al. Cell Chem. Biol. (2020).

Article Snippet: Most cIAP inhibitors are mimetics of second mitochondria-derived activator of caspases (SMAC).

Techniques: