Journal: Nature communications

Article Title: CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity.

doi: 10.1038/s41467-022-29469-0

Figure Lengend Snippet: Fig. 2 CHMP2A-KO increases GSC and HNSCC cell lines sensitivity to NK cell-mediated cytotoxicity. a 4 hour cytotoxicity assay to determine the effect of CHMP2A in GSC resistance to NK cell-mediated cytotoxicity. Two independent sgRNAs were used (sg#2, sg#3). b immunoblot analysis of WT and KO GSC lines KO for CHMP2A using two sgRNAs (sg#2, sg#3). GAPDH serves as a loading control. c 4 hour cytotoxicity assay to determine the effect of CHMP2A in HNSCC-resistance to NK cell-mediated cytotoxicity. d Western blot analysis showing the rescue of KO phenotype. Here we show Cal27 cells expressing WT levels of CHMP2A, no CHMP2A (KO), or complemented CHMP2A (KO+ overexpressed CHMP2A). HEK293 CHMP2A overexpression lysate (OL) was used as a control. The overexpressed CHMP2A is fused with DDK and MYC tags explaining the 4 kDa increase in molecular weight. For this western blot, GAPDH was used as a loading control. e 4 hour cytotoxicity assay to determine the effect of CHMP2A in Cal27 cells expressing WT levels of CHMP2A, no CHMP2A (KO), or complemented CHMP2A (KO+ overexpressed CHMP2A). a, c, e The error bars represent standard error from the mean (±SEM) across n = 3 replicates. Statistical analysis was performed by one-tailed paired Student t test. Representative of n = 3 independent experiments.

Article Snippet: In all, 8 × 105 cells Cal27-KO were transfected with CHMP2A (NM_014453) Human Tagged ORF Clone (Origene) via nucleofection using Amaxa 2D (Lonza) and Cell Line Nucleofector Kit V (Lonza) following manufacturer’s protocol.

Techniques: Cytotoxicity Assay, Western Blot, Control, Expressing, Over Expression, Molecular Weight, One-tailed Test

Journal: Nature communications

Article Title: CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity.

doi: 10.1038/s41467-022-29469-0

Figure Lengend Snippet: Fig. 3 Gene expression analysis of CHMP2A-KO in GSCs. a Differential gene expression comparing CHMP2A-KO vs shCONT (n = 2 samples per cell line). Genes in red are upregulated with a knockout at log2 FC>1 and FDR<0.05, whereas genes in blue are downregulated with a knockout at log2 FC < −1 and FDR < 0.05. Top altered genes are labeled in the panel. b Reactome and KEGG gene set enrichment analysis of genes upregulated with CHMP2A KO. Y axis indicates −log10 FDR of the gene set enrichment.

Article Snippet: In all, 8 × 105 cells Cal27-KO were transfected with CHMP2A (NM_014453) Human Tagged ORF Clone (Origene) via nucleofection using Amaxa 2D (Lonza) and Cell Line Nucleofector Kit V (Lonza) following manufacturer’s protocol.

Techniques: Gene Expression, Knock-Out, Labeling

Journal: Nature communications

Article Title: CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity.

doi: 10.1038/s41467-022-29469-0

Figure Lengend Snippet: Fig. 4 Secretion of CXCL10 and CXCL12 from CHMP2A-KO tumor cells increases NK cell migration. a ELISA comparing CXCL10 concentration WT and KO in CW468 and Cal27 cells. The error bars represent ±SEM across n = 3 independent experiments two-tailed Student’s t test was performed to determine statistical significance. b ELISA comparing CXCL10 concentration in WT and KO in CW468 and Cal27 cells. Error bars represent ±SEM from the mean across n = 3 independent experiments two-tailed Student’s t test was performed to determine statistical significance. c charts representing the number of NK cells migrating towards CW468-WT and KO (top chart) and Cal27-WT and KO (lower chart) cells analyzed by flow cytometry. Error bars represent ±SEM across n = 3 independent experiments. Two-tailed Student’s t test was performed to determine statistical significance. d Immunoblot analysis of NF-κB P65 in Cal27 CHMP2A-WT or KO. GAPDH has been used as a loading control. e Dual-luciferase reporter assay showing NF-κB activity in Cal27 CHMP2A-WT and KO cells. Error bars represent ±SEM from the mean across n = 3 replicates, two-tailed Student’s t test was performed to determine statistical significance. d, e Representative of n = 3 independent experiments.

Article Snippet: In all, 8 × 105 cells Cal27-KO were transfected with CHMP2A (NM_014453) Human Tagged ORF Clone (Origene) via nucleofection using Amaxa 2D (Lonza) and Cell Line Nucleofector Kit V (Lonza) following manufacturer’s protocol.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Concentration Assay, Two Tailed Test, Cytometry, Western Blot, Control, Luciferase, Reporter Assay, Activity Assay

Journal: Nature communications

Article Title: CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity.

doi: 10.1038/s41467-022-29469-0

Figure Lengend Snippet: Fig. 5 Cal27-derived EVs secretion is reduced in CHMP2A-KO cells and in tipifarnib treated WT Cal27 cells. a, b Charts showing the size distribution and number of EVs secreted from Cal27-WT (a) and KO (b) cells. Samples were loaded on a Nanosight LM10 and analyzed for 1 min for each of n = 5 technical replicates and the error bars represent ±SEM across n = 5. c comparison of EV number in Cal27-WT and KO calculated on 10 μl of EV suspension diluted in 1 ml of DPBS. d average EVs size analyzed during nanoparticle tracking of Cal27-WT and KO derived EVs. e comparison of EVs number in Cal27- WT treated with tipifarnib (Tip) and the corresponding DMSO control (CTRL) calculated on 10 μl of EVs suspension diluted in 1 ml of DPBS. c–e error bars represent ±SEM across n = 5 technical replicates and unpaired two-tailed Student’s t test was performed to determine statistically. f 4 hour cytotoxicity assay using NK cells as effectors against Cal27-WT, KO treated with tipifarnib or DMSO (CTRL). Error bars represent ±SEM, n = 3 replicates. Statistical analysis was performed by two-way ANOVA and Bonferroni’s post hoc multiple comparison test (comparing Cal27-WT vs Cal27-WT + Tip E:T, 10:1 p < 0.0001; 5:1 p < 0.0001; 2:1 and 1:1 ns). Data are shown in a–f, representative of n = 3 independent experiments.

Article Snippet: In all, 8 × 105 cells Cal27-KO were transfected with CHMP2A (NM_014453) Human Tagged ORF Clone (Origene) via nucleofection using Amaxa 2D (Lonza) and Cell Line Nucleofector Kit V (Lonza) following manufacturer’s protocol.

Techniques: Derivative Assay, Comparison, Suspension, Control, Two Tailed Test, Cytotoxicity Assay

Journal: Nature communications

Article Title: CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity.

doi: 10.1038/s41467-022-29469-0

Figure Lengend Snippet: Fig. 7 CHMP2A-KO HNSCC xenograft model shows increased sensitivity to NK cells. a Schematic of in vivo treatment. Non-obese diabetic/severe combined immunodeficiency/γc−/−(NSG) mice were inoculated subcutaneously with 6 × 106 cells (Cal27-WT or KO) and injected i.v. with 1 × 107 NK cells. NK cells were supported by injections IL-15 daily and IL-2 every other day for 1 week. Tumor volume was monitored every 2–3 days. b Tumor volume progression over 21 days shown as mean ±SEM of n = 5 mice per treatment. Statistical analysis was performed by two-way ANOVA and Bonferroni’s post hoc multiple comparison test (ns, not significant). c Representative flow cytometry dot plots showing increased NK cell infiltration in Cal27 CHMP2A KO tumors. The gating strategy has been defined by combining NK cells and a cell suspension from the untreated tumor. Tumor samples without NK cell treatment were mixed with 2 × 104 human NK cells, stained with anti-human CD45 and anti-human CD56 antibodies or isotype controls, and analyzed. Cells double positive for (human) hCD45 and hCD56 were gated and the gate was used to quantify the number of infiltrating NK cells in Cal27-WT and CHMP2A KO tumors. The graph shows the average NK cell infiltration for all mice in each group (n = 4 mice per group). Error bars represent ±SEM across n = 4 technical replicates and an unpaired one-tailed Student’s t test was performed to determine statistical significance. d Cartoon describing the proposed mechanism of CHMP2A KO in tumor cells. Tumor cells secrete EVs-bearing ligands like MICA/B and TRAIL, which suppress the antitumor effect of NK cells by binding to NKG2D and TRAIL-R to induce apoptosis on NK cells. By CHMP2A KO we reduce EVs secretion from tumor cells decreasing their inhibitory function. Tumor cells CHMP2A KO also secrete CXCL10 and CXCL12, chemokines that increase NK cell migration culminating in enhanced NK cell-mediated cytotoxicity (the cartoon has been created with Biorender.com).

Article Snippet: In all, 8 × 105 cells Cal27-KO were transfected with CHMP2A (NM_014453) Human Tagged ORF Clone (Origene) via nucleofection using Amaxa 2D (Lonza) and Cell Line Nucleofector Kit V (Lonza) following manufacturer’s protocol.

Techniques: In Vivo, Injection, Comparison, Cytometry, Suspension, Staining, One-tailed Test, Binding Assay, Migration

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Matrix Metalloproteinase-19 is Highly Expressed in Astroglial Tumors and Promotes Invasion of Glioma Cells

doi: 10.1097/nen.0b013e3181ce9f67

Figure Lengend Snippet: FIGURE 4. Silencing of matrix metalloproteinase 19 (MMP19) reduces glioblastoma (GB) cell invasion in Transwell migration assays and into brain tissue sections. (A) U343 GB cells were transfected with no (control), control siRNA (C-Si), a nonrelated siRNA (C-n.r.; 250 Kmol/L siRNA against seprase, a serine protease), or with different concentrations MMP19-specific siRNA (Si-MP19). The MMP19 mRNA reduction was determined by quantitative reverse transcription polymerase chain reaction (calculated by the $$ CT method, n = 3 T SD). For subsequent assays, 250-Kmol/L transfected cells that showed MMP19 reduction to 17% were used. (B) In Boyden chamber assays through 5-Hm porous membranes, silenced and control cells migrated at similar rates when the chambers were uncoated (none); MMP19-silenced cells migrated more slowly through laminin-coated and somewhat more slowly through Matrigel- or brevican-coated membranes after 90 minutes (n = 15 for controls and Matrigel, n = 5 for others; mean T SD; *p G 0.05, ***p G 0.001); control siRNA-transfected cells behaved similar to control cells (not shown). (C) Invasion of MMP19-silenced U343 GB cells into sections of brain tissue is reduced compared with most controls. Transfected GB cells were either green (MMP19 siRNA) or red (control siRNA) fluorescence labeled; mixtures of equal numbers of both cells were applied on brain tissue slices. At 48 or 72 hours, the sections were fixed and inspected with an inverse microscope with apotome from the bottom (example: middle); red and green fluorescent cells were counted in the same areas and depths. Cell counts from 4 different experiments with individual cells/tissues are shown (right).

Article Snippet: Cell migration was monitored as described ([21] usually after 90 minutes) in 48-well Boyden-chambers (Neuro Probe, Baltimore, MD) using polycarbonate membranes with 5-Km pores that were uncoated (basal migration) or coated with Matrigel (a basement membrane protein mixture secreted by EHS mouse sarcoma cells containing laminin, collagen IV, heparan sulfate proteoglycans, and entactin), laminin (both from BD Biosciences, San Jose, CA), or human recombinant brevican (a member of the lectican family of chondroitin sulfate proteoglycans that is predominantly expressed in the CNS and is a major brain ECM component), and human recombinant glycosylated Asp23-Pro911, C-terminal His-tagged expressed in mouse myeloma cells (4009-BC; R&D Systems, Minneapolis, MN).

Techniques: Migration, Transfection, Control, Reverse Transcription, Polymerase Chain Reaction, Labeling, Microscopy

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Matrix Metalloproteinase-19 is Highly Expressed in Astroglial Tumors and Promotes Invasion of Glioma Cells

doi: 10.1097/nen.0b013e3181ce9f67

Figure Lengend Snippet: FIGURE 5. Matrix metalloproteinase 19 (MMP19) degrades brevican. Human recombinant brevican (1, 1 Kg; 2 and 3, 3 Kg) was incubated at 37-C at pH 7.4 with no MMP19 (1, control), MMP19 inactive mutant (2, MMP19EA), or wild-type MMP19 (3, MMP19 WT). Aliquots were withdrawn after different times (0, 10, 24, 34 hours) and examined by immunoblotting. The intensity of the 160-kd brevican band diminishes, and several fragments appear in a time-dependent manner in the MMP19WT lanes.

Article Snippet: Cell migration was monitored as described ([21] usually after 90 minutes) in 48-well Boyden-chambers (Neuro Probe, Baltimore, MD) using polycarbonate membranes with 5-Km pores that were uncoated (basal migration) or coated with Matrigel (a basement membrane protein mixture secreted by EHS mouse sarcoma cells containing laminin, collagen IV, heparan sulfate proteoglycans, and entactin), laminin (both from BD Biosciences, San Jose, CA), or human recombinant brevican (a member of the lectican family of chondroitin sulfate proteoglycans that is predominantly expressed in the CNS and is a major brain ECM component), and human recombinant glycosylated Asp23-Pro911, C-terminal His-tagged expressed in mouse myeloma cells (4009-BC; R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Incubation, Control, Mutagenesis, Western Blot

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Structure and specificity of binding of chimeric BCMA×PDL1 bsAb. ( A ) Starting from the N-terminus, the fused heavy chain is composed of the anti-BCMA J22.9 antibody VH sequence-CH1 hinge1-A1linker-anti-PDL1 atezolizumab VH sequence-CH1 hinge2-CH2-CH3. The CH1 hinge-CH2 and CH3 sequences are from human IgG1. The two light chains (both k) are anti-BCMA J22.9 VL-CL and anti-PDL1 atezolizumab VL-CL. The red dot indicates the paired complementary mutations on CH1 and CL of the anti-PDL1 moiety to drive correct light chain pairing . ( B ) Specificity of binding of the purified bsAb and respective mAbs was tested by flow cytometry on mBCMA + and PDL1 + single-positive cell lines, KMS11 and HDLM2, respectively. MFI: mean fluorescence intensity. %: percentage of mBCMA-positive cells.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay, Sequencing, Purification, Flow Cytometry, Fluorescence

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Relative binding affinity of BCMA×PDL1 bsAb and respective mAbs for target antigens. The relative affinity of the BCMA×PDL1 bsAb, anti-BCMA, and anti-PDL1 mAbs was tested by flow cytometry, using increasing concentrations of primary antibodies and detection with anti-human Fc-FITC secondary antibody. ( A ) Binding of bsAb and mAbs to mBCMA + KMS11 cell line. ( B ) Binding of bsAb and mAbs to PDL1 + HDM2 cell line. ( C ) Relative binding affinities (IC 50 ) for each antigen; NA: not applicable.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay, Flow Cytometry

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Binding constants determined by SPR studies.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Surface plasmon resonance analysis. The sensorgrams shown were obtained by injecting recPDL1 or recBCMA, alone ( A – C ) or in succession ( D ) over immobilized anti-PDL1 ( A ), anti-BCMA ( B ), or BCMA×PDL1 bsAb ( C , D ). The antigens were flowed for 3 min, as indicated by the dashed lines.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: SPR Assay

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: The BCMA×PDL1 bsAb blocks APRIL binding to mBCMA and PD1-PDL1 interaction. ( A ) To test the ability of bsAb to block APRIL binding to mBCMA, we used CEM-mBCMA + cell line, increasing concentrations of bsAb and anti-BCMA mAb, a Flag-tagged APRIL protein, and an anti-Flag antibody. *: p < 0.05. ( B ) For assessing the inhibition of the PD1-PDL1 axis, we tested increasing concentrations of BCMA×PDL1 bsAb or anti-PDL1 mAb in the cell-based PD1/PDL1 Blockade Bioassay. ( C ) Cell-based PD1/PDL1 Blockade Bioassay in presence of recBCMA. Atezolizumab and cetuximab were used as positive and negative controls, respectively. *: p < 0.05 vs. PDL1.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay, Blocking Assay, Inhibition, Bioassay

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: The BCMA×PDL1 bsAb mediates CDC of mBCMA + cells. ( A ) The BJAB-mBCMA + cell line was incubated with increasing concentrations of bsAb, mAbs, or RTX as positive control and in the presence of 50% HS as a source of complement. CDC was measured after 4 h by 7-AAD staining and flow cytometry. *: p < 0.05 and **: p < 0.01. ( B ) Flow cytometry histograms showing the expression of mBCMA of BJAB cells stably expressing BCMA in presence or absence of DAPT. MFI: mean fluorescence intensity. %: percentage of mBCMA-positive cells.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Incubation, Positive Control, Staining, Flow Cytometry, Expressing, Stable Transfection, Fluorescence

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. Apoptosis was induced in HeLa and immortalized MEFs using STS for the indicated times, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced in HeLa, BT-549 and COS-7 cells with etoposide. NT (No Treatment). In primary MEFs apoptosis was induced with 100 µM etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 µM of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and with 1.75 µM STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Western Blot, Transfection

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: AI. HeLa ARTS knockdown (ARTS KD) cells and Sept4/ARTS KO MEFs show significantly higher levels of steady-state Bcl-2 protein when compared with WT cells. This suggests that ARTS plays an important role in regulating Bcl-2 levels. B. WT and ARTS KD HeLa cells were treated with 1.75 µM STS. Western Blot analyses demonstrate that while decreased Bcl-2 levels were seen in apoptotic WT HeLa cells, Bcl-2 levels in ARTS KD HeLa cells remained unchanged. C. Western Blot analyses of cytosolic fractions of BT-549, HeLa WT and HeLa ARTS KD cells reveal that endogenous Bcl-2 is found in the cytosol of WT STS-treated cells. In contrast, a strong inhibition in translocation of Bcl-2 to the cytosol was seen in ARTS KD HeLa cells. This suggests that ARTS is required for the proper translocation of Bcl-2 from mitochondria to the cytosol upon apoptotic induction. D. Immuno-fluorescence (IF) was performed on HeLa and a stable Bcl-2 knockdown (Bcl-2 KD) cells. The fraction of cells with cytosolic staining of ARTS is represented in the bar chart. While only a small portion of WT untreated (NT) HeLa cells show the presence of ARTS in the cytosol, a significant increase in cells containing cytosolic ARTS was seen following STS treatment. In contrast, the majority of HeLa Bcl-2 KD NT cells exhibit cytosolic ARTS (four fold higher than WT HeLa cells), and, only a slight increase in cells with cytosolic ARTS is seen after STS treatment. (* * p-value ≤ 0.01). See also supplemental Figure S1. E. Cytosolic and mitochondrial fractions of WT MEFs and Bcl-2 KO MEFs were analyzed by WB analysis with COX IV as a mitochondrial and GAPDH as a cytosolic marker. In Bcl-2 KO MEFs, the majority of ARTS was in the cytosol. This suggests that Bcl-2 is involved in localizing ARTS to mitochondria. F. IF of WT MEFs and Sept4/ARTS KO MEFs transiently transfected with GFP-Bcl-2. Cellular localization of Bcl-2 was quantified and the fraction of cells with cytosolic Bcl-2 is shown in the bar charts. While a significant increase in cytosolic Bcl-2 was seen in apoptotic WT MEFs, the levels of cytosolic Bcl-2 in Sept4/ARTS KO MEFs remained unchanged. See also supplemental Figure S1. G. Subcellular fractionation of HeLa cells was followed by in vivo ubiquitylation of each fraction. IgG represents the control cells incubated with non-specific IgG. Poly-ubiquitylated forms of Bcl-2 were seen only in the cytosolic fraction. * Represents the IG heavy chain. This indicates that ubiquitylation of Bcl-2 occurs in the cytosol and that ARTS is required for translocation of Bcl-2 to the cytosol during apoptosis.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Western Blot, Inhibition, Translocation Assay, Fluorescence, Staining, Marker, Transfection, Fractionation, In Vivo, Incubation

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: AI. IP of ARTS in COS-7 cells transiently transfected with 6-Myc-ARTS using a monoclonal anti-ARTS antibody (Sigma-Aldrich). Endogenous ARTS was immunoprecipitated from HeLa and BT-549 cells. Western blot analyses show that in COS-7, HeLa and BT-549 cells, Bcl-2 and XIAP co- precipitate with ARTS. AII. HeLa cells were treated with 1.75 µM STS and IP of ARTS was performed as described in AI. While binding of ARTS to Bcl-2 was seen in non-treated cells (NT), binding of ARTS to XIAP and Bcl-2 is increased in STS treated cells. B. Recombinant His-ARTS, Bcl-2 and GST-XIAP were incubated overnight at 4 °C. Pulldown of GST-XIAP shows that binding of Bcl-2 to XIAP depends on ARTS. C. To assess proximity of proteins, consistent with complex formation, we used BiFC. HeLa cells were transiently transfected with ARTS, Bcl-2 and XIAP fused to parts of YFP YFP-Venus. Jun and bFos, known to form heterodimers, served as a positive control (p.c.). Jun and bFosdelZIP lacking the carboxyl-terminal half of the bFos were used as a negative control (n.c.). The fluorescent signal indicating the proximity of each pair of proteins was measured by flow cytometry. Mean fluorescence intensity (MFI). FACS results were normalized to the readings of transfection efficiency reporter (pdsRED). The values represent mean values ± SE of three independent experiments (*, p ≤ 0.05; **, p ≤ 0.01). The Y-axis represents the ratio between YFP fluorescence (reflecting binding of a pair of proteins) and the red fluorescence (marking the transfected cells). FACS analyses reveal that ARTS can bind to both Bcl-2 and to XIAP. Yet, only background levels of fluorescence were seen with XIAP and Bcl2, suggesting that these two proteins do not bind each other. These results indicate that ARTS, XIAP and Bcl-2 form a ternary complex, and that ARTS is required for the formation of this complex. See also supplemental Figure S2.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Recombinant, Incubation, Positive Control, Negative Control, Flow Cytometry, Fluorescence

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. A custom designed Bcl-2 derived peptide array (CelluSpot™) was incubated with recombinant (His)6-Lipo-TEV (HLT-) tagged ARTS. The peptide array was also incubated with recombinant HLT-tag alone which served as a negative control. The arrays were probed with the indicated antibodies. Lower panel depicts the Bcl-2 peptides which bind to ARTS. These peptides are schematically represented across the Bcl-2 domains; the black lines indicate strong binding and the grey lines a weak to moderate binding. B. Bcl-2 expression vectors; full length and four constructs deleting each BH domain in Bcl-2 are illustrated at the top (FLD- Flexible Loop Domain, TM-Transmembrane domain). These vectors were co-transfected with 6myc-ARTS into Bcl-2 KO MEFs. IP of ARTS was performed in MEFs transfected with Bcl-2-BH deletions, WT Bcl-2 (positive control) and empty vector (negative control). A significant reduction in binding of ARTS to Bcl-2delBH3 was seen. See also supplemental Tables S1 and S2. C. BIFC assays were performed as described in Figure 3C. HeLa cells were co-transfected with vectors expressing ARTS, Bcl-2 and Bcl-2delBH3 fused to either VN or VC parts of the YFP Venus fragment (ARTS-VN, Bcl-2-VC. Bcl-2delBH3 –VC). A 47% decrease in the proximity between ARTS and Bcl-2delBH3 was seen compared to WT Bcl-2. Jun/bFos served as positive control (p.c.), and Jun/bFosdel ZIP as negative control (n.c.). Results are shown as mean + S.E. of three independent experiments. (*, p ≤ 0.05; **, p ≤ 0.01). See also supplemental Figure S3. D. In vivo ubiquitylation of Bcl-2 WT and Bcl-2delBH3. MEFs Bcl-2 KO cells were transfected with Flag-Bcl-2WT or Flag-Bcl-2delBH3. Cells were treated with MG132 and STS for one hour or left untreated. Proteins were immunoprecipitated with anti-ubiquitin. Poly-ubiquitylated forms of Bcl-2 were detected using an anti-Bcl-2 antibody. While WT Bcl-2 undergoes ubiquitylation following treatment with STS, no ubiquitylation of Bcl-2delBH3 was seen. See also supplemental Figure S4.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Derivative Assay, Peptide Microarray, Incubation, Recombinant, Negative Control, Binding Assay, Expressing, Construct, Transfection, Positive Control, Plasmid Preparation, In Vivo, Immunoprecipitation

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: AI. HeLa cells were transiently transfected with Bcl-2 or co-transfected with Bcl-2 and XIAP or with Bcl-2 and XIAPdelRing. AII. MEFs were produced from WT mice and from XIAPdelRing 14-day old mouse embryos. WB and densitometry analyses reveal a significant accumulation of Bcl-2 in HeLa cells co-transfected with XIAPdelRing and in XIAPdelRing MEFs. B. Sept4/ARTS KO MEFs were transfected with WT ARTS and a mutant version of ARTS lacking its unique C-terminus (Cdel-ARTS) which cannot bind to XIAP. Sept4/ARTS KO MEFs transfected with WTARTS exhibit decreased levels of Bcl-2 following apoptotic induction (UV), while MEFs transfected with the Cdel-ARTS have increased levels of Bcl-2. C. In vitro ubiquitylation assays were performed by incubating recombinant Bcl-2 with recombinant XIAP, E1, E2-UbcH5b and ubiquitin. In the control reactions the indicated components were excluded. Ubiquitylation of Bcl-2 was seen only upon addition of XIAP. D. In vitro ubiquitylation assays with purified Bcl-2 and recombinant XIAP, cIAP1, Parkin, or Siah2. Only addition of XIAP resulted in the ubiquitylation of Bcl-2. See also Supplemental Figure S5 E. In vivo ubiquitylation in WT and XIAPdelRing MEFs. MEFs were co-transfected with ARTS, Bcl-2 and HA-ubiquitin. Cells were treated with 20 µM MG-132 for 6h and concomitantly treated with 1.75 µM STS and IP was performed with an anti-Bcl-2 antibody. *Represents the heavy chain of the antibody. While significant ubiquitylation of Bcl-2 occurred after treatment with STS in WT MEFs, no ubiquitylation of Bcl-2 was seen in XIAPdelRing MEFs. F. In vivo ubiquitylation assays using WT and XIAP KO MEFs. No ubiquitylation of Bcl-2 was seen in XIAP KO MEFs. Collectively these results show that XIAP serves as the specific E3-ligase for Bcl-2 and is required for its degradation.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Transfection, Produced, Mutagenesis, In Vitro, Recombinant, Purification, In Vivo

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. In vitro ubiquitylation assays were performed by incubating recombinant Bcl-2 with XIAP and ARTS, E1, E2-UbcH5b and ubiquitin in the presence of ATP at 37 °C for 1 hr. A significant increase in the appearance of ubiquitylated forms of Bcl-2 was seen in the presence of ARTS. B. HeLa WT or HeLa ARTS KD cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and STS for 0.5 h. Bcl-2-ubiquitin conjugates were seen in the apoptotic WT HeLa cells, but not in ARTS KD HeLa cells. C. In vivo ubiquitylation assays of Sept4/ARTS KO MEFs transfected with either empty vector or ARTS expression vector, followed by treatment with MG132 and STS for 3 h. *Represents the IgG heavy chain. While no ubiquitylation of Bcl-2 was observed in the Sept4/ARTS KO MEFs, transfection of ARTS restored their ability to generate poly-ubiquitylated forms of Bcl-2 upon induction of apoptosis.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: In Vitro, Recombinant, Transfection, In Vivo, Plasmid Preparation, Expressing

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. Schematic representation of Bcl-2 highlighting its four lysine residues. B. MS/MS spectrum spanning the ubiquitylation site in Bcl-2. The trypsin cleaved peptides were enriched for Gly-Gly peptides using Pilot Ubiquitin Remnant Motif (K-ε-GG) kit and subjected to liquid chromatography-mass spectrometry separation. Precursor ion (brown) represents the uncleaved GFP-Bcl-2. Lines represent the relative abundance of detected peptides. The lines in black represent peptides that are not cleaved products of the precursor ion (y1–y6) and precursor ion of the un-fragmented peptide (brown). Lines in green (y1–y8) represent the cleaved GFP-Bcl-2 peptides and their corresponding amino acid sequence. KGG (red) represents an ubiquitylated lysine. This analysis identified Lysine 17 in Bcl-2 as the acceptor for XIAP-mediated ubiquitylation. C. MEFs Bcl-2 KO and HeLa cells were transfected with expression vectors for WT Bcl-2 (WT), Bcl-2 containing a substitution mutation of Lysine 17 into Alanine (K17A), and Bcl-2 in which all Lysines were changed to Alanine (No K). Increased levels of mutant Bcl-2 (K17A, No K) were seen upon apoptotic induction. This was accompanied by a decrease in apoptosis, as shown with three different apoptotic markers. Densitometry analyses are shown in the lower panel. D. Bcl-2 KO MEFs and HeLa cells were transfected with Bcl-2 as in (C) together with a GFP-cleaved caspase-3 reporter. Bcl-2 KO MEFs and HeLa cells expressing lysine mutants had significantly less cleaved caspase 3-positive cells compared to WT Bcl-2. These results suggest that Lysine 17 is the main acceptor for ubiquitylation of Bcl-2.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Tandem Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Sequencing, Transfection, Expressing, Mutagenesis