Journal: Oncotarget

Article Title: Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin

doi: 10.18632/oncotarget.13975

Figure Lengend Snippet: ( A ) Meprin α expression was analyzed by qRT-PCR in HuH7 cells following transfection of M1 or M2 meprin a siRNAs ( p = 0.0002, n =3). ( B ) Same experiment in Hep3B cells ( p = 0.0002, n =3). ( C ) Western blot of HuH7 extracts showing decreased expression of meprin α following transfection with a control siRNA (C) or with the M1 or M2 siRNAs. ( D ) Overexpression of meprin α with a V5 tag in HuH7 or Hep3B cells. Stable cell lines were established as described in Methods. ( E ) Immunofluorescence for Meprin (left) or the V5 tag (right). ( F ) Meprin α proteolytic activity in the conditioned medium is expectedly increased in HuH7 cells overexpressing meprin-V5.

Article Snippet: Quantitative RT-PCR was performed using predesigned TaqMan probes (Hs00194410_m1, Hs00195535_m1, and -Hs00272632_m1 for MEP1A, MEP1B, and RUVBL2 (Reptin), respectively) and the ABI BioMark HD reader (Fluidigm).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Over Expression, Stable Transfection, Immunofluorescence, Activity Assay

Journal: Oncotarget

Article Title: Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin

doi: 10.18632/oncotarget.13975

Figure Lengend Snippet: ( A ) HuH7 cell proliferation was assessed by direct cell counting in cells transfected with a control siRNA, or siRNAs targeting Reptin or meprin α (M1 and M2). Reptin silencing significantly decreased proliferation. The M2 siRNA had no significant effect whereas the M1 siRNA has a slight but significant effect ( p = 0.05, n = 3). ( B ) MTS assay in HuH7 cells showing no significant effect of the two meprin siRNAs on proliferation. ( C ) Cell counting experiment in Hep3B cells showing that Reptin, but not meprin silencing, reduces proliferation ( n = 3). ( D ) Growth of HuH7 (left) or Hep3B cells (right) overexpressing meprin-V5 assessed with a MTS assay ( n = 3). ( E ) Growth of HuH7 (left) or Hep3B cells (right) treated with 20 nM recombinant meprin α assessed with a MTS assay ( n = 3).

Article Snippet: Quantitative RT-PCR was performed using predesigned TaqMan probes (Hs00194410_m1, Hs00195535_m1, and -Hs00272632_m1 for MEP1A, MEP1B, and RUVBL2 (Reptin), respectively) and the ABI BioMark HD reader (Fluidigm).

Techniques: Cell Counting, Transfection, Control, MTS Assay, Recombinant

Journal: Nature Communications

Article Title: Co-translational determination of quaternary structures in chaperone factories

doi: 10.1038/s41467-026-68687-8

Figure Lengend Snippet: A Schematic of the HSP90/R2TP chaperone. DII is the domain II of RUVBL1 and RUVBL2. Created in BioRender, Tartier, S. (2026) https://BioRender.com/5ulccbz . B Schematic of the cassette integrated at the RUVBL1 genomic locus and micrographs of HCT116 cells with a RUVBL1-GFP allele and imaged by fluorescent microscopy. Green: RUVBL1-GFP. Blue: DAPI staining. Scale bar: 5 µm. C Volcano plot of the RUVBL1-GFP RIP-seq experiment, showing the pValue (y axis; -Log10) as a function of the enrichment in the GFP IP over the control IP (x axis; Log2 (GFP IP/control IP)). Each dot represents an mRNA that is colored according to the code on the right of panel E. The statistical test was a two-sided QL F-test. D Volcano plot of the RUVBL1-GFP RIP-seq experiment in cells treated with puromycin for 1 h. Legend as in ( C ). E Volcano plot comparing the RUVBL1-GFP RIP-seq in untreated versus CB-6644-treated condition (16 h). The graph shows the p-value (y axis; -Log10) as a function of the fold change in the GFP IPs of treated versus non-treated cells (x axis; Log2(GFP IP/control IP)). The statistical test was a two-sided QL F-test.

Article Snippet: Membranes were blocked in PBS with 10% (w/v) milk and 0.05% Tween-20 (Euromedex) before incubation with primary antibodies diluted 1:1000 in PBS 0.05% Tween overnight at 4 ° C. After washing with 1XPBS/0.05% Tween-20, membranes were incubated with primary antibodies anti-RUVBL2 (Proteintech 10195-1-AP, 1/1000 dilution) or anti-RPS3 (Proteintech 66046-1-Ig, 1/1000 dilution) and secondary antibodies coupled to HRP (1/5000 dilution) and visualized using SuperSignal West Femto or Pico (Thermo Scientific) with LAS 4000 mini (GE Healthcare).

Techniques: Microscopy, Staining, Control

Journal: Advanced Science

Article Title: Epigenetic Control of Translation Checkpoint and Tumor Progression via RUVBL1‐EEF1A1 Axis

doi: 10.1002/advs.202206584

Figure Lengend Snippet: RUVBL1 controls MYC chromatin localization and transactivation activity in EwS. A) RNAseq and GSEA analyses showing changes in expression of the MYC upregulated target gene set in sgCtrl and sgRUVBL1 transduced (day 5) A673‐Cas9 cells (two independent sgRNA sequences per group). (Right) Each dot indicates one gene set from the GSEA Molecular Signature Database (MSigDB; total 238 gene sets from the Hallmark and Oncogenic Signature [C6] collections). NES: Normalized enrichment score. B) Growth competition assay of sgCtrl (gray lines; n = 2 independent sgRNA sequences) and sgMYC (purple lines; n = 5 independent sgRNA sequences) in A673‐Cas9 cells. C) Cell cycle monitored by EdU incorporation, and D) cellular apoptosis detected by active caspase 3 + /DAPI − in A673‐Cas9 cells transduced with sgCtrl and sgMYC ( n = 3 for each group). E) Western blot of RUVBL1, MYC, histone H4, and GAPDH in A673‐Cas9 cells transduced with sgCtrl and sgRUVBL1 (two independent sgRNA sequences per group). F) Co‐IP of RUVBL1 (flag‐tagged) with RUVBL2 and MYC in HEK293 cells. G) Meta plots (top) and heatmaps (bottom) showing ChIP‐seq signal of MYC at TSSs ± 3 kb regions for all genes in A673‐Cas9 cells transduced with sgCtrl and sgRUVBL1. H) Profiles of MYC ChIP‐seq and (I) ChIP‐qPCR at RUVBL1 locus in A673‐Cas9 cells transduced with sgCtrl and sgRUVBL1 ( n = 3 for each group). J) RT‐qPCR of RUVBL1 mRNA in A673‐Cas9 cells transduced with sgCtrl and sgMYC ( n = 3 for each group). K) Western blot of MYC, RUVBL1, and GAPDH in A673‐Cas9 cells transduced with sgMYC (two independent sgRNA sequences per group). L) Model of a feed‐forward network between RUVBL1 and MYC in EwS. Data are represented as mean ± SEM. * P < 0.01 compared to sgCtrl by two‐sided Student's t ‐test.

Article Snippet: PVDF membranes were immersed in 5% nonfat milk then incubated at 4 °C overnight with primary antibodies against RUVBL1 (HPA019947, Sigma; 1:1000), RUVBL2 (12668S, Cell Signaling Technology; 1:1000), MYC (13987S, Cell Signaling Technology; 1:1000), EEF1A1 (PA5‐17213, Thermo Fisher; 1:1000), KAT5 (12058S, Cell Signaling Technology; 1:1000), H4K8ac (07‐328, Millipore; 1:1000), H4K12ac (61527, Active Motif; 1:1000), histone H4 (ab177840, Abcam; 1:1000), GAPDH (2118S, Cell Signaling Technology; 1:5000), flag‐tag (F7425, Millipore; 1:5000), and V5‐tag (13202S, Cell Signaling Technology; 1:1000).

Techniques: Activity Assay, Expressing, Competitive Binding Assay, Transduction, Western Blot, Co-Immunoprecipitation Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Epigenetic Control of Translation Checkpoint and Tumor Progression via RUVBL1‐EEF1A1 Axis

doi: 10.1002/advs.202206584

Figure Lengend Snippet: Lysine 108 in RUVBL1 is required for the interaction between RUVBL1 and MYC. A) Schematic outline of RUVBL1 high‐density CRISPR gene body scan in A673‐Cas9 cells. B) 2D annotation of RUVBL1 CRISPR scan. The gray line indicates the smoothened model of the CRISPR scan score derived from 194 sgRNAs (dots) targeting the coding exons of RUVBL1 ( n = 3 replicates). The median CRISPR scan scores of the positive control (red line; defined as −1.0) and negative control (blue line; defined as 0.0) sgRNAs are highlighted. C) 3D annotation RUVBL1 CRISPR scan score relative to a cryo‐EM structural model of a hexamer consists of three RUVBL1 and three RUVBL2 proteins (PDB ID: 5OAF). D) Western blot showing doxycycline (DOX)‐induced expression of flag‐tagged WT‐ and K108A‐RUVBL1 in A673‐dCas9‐KRAB cells. E) Effect of WT‐ and K108A‐RUVBL1 expression on the growth competition assay of A673‐dCas9‐KRAB cells transduced with sgiCtrl and sgiRUVBL1 ( n = 3 for each group). F) Western blot of RUVBL1, H4K8ac, H4K12ac, EEF1A1, histone H4, and GAPDH in WT‐ and K108A‐RUVBL1 expressing A673‐dCas9‐KRAB cells transduced with sgiCtrl and sgiRUVBL1. G) Co‐IP of WT‐ and K108A‐RUVBL1 (flag‐tagged) with RUVBL2, KAT5, and MYC in HEK293 cells. H) Model of RUVBL1 supporting MYC chromatin binding and target gene expression. Data are represented as mean ± SEM. * P < 0.01 compared to sgCtrl by two‐sided Student's t ‐test. Source data are available for this figure: SourceData F5 B.

Article Snippet: PVDF membranes were immersed in 5% nonfat milk then incubated at 4 °C overnight with primary antibodies against RUVBL1 (HPA019947, Sigma; 1:1000), RUVBL2 (12668S, Cell Signaling Technology; 1:1000), MYC (13987S, Cell Signaling Technology; 1:1000), EEF1A1 (PA5‐17213, Thermo Fisher; 1:1000), KAT5 (12058S, Cell Signaling Technology; 1:1000), H4K8ac (07‐328, Millipore; 1:1000), H4K12ac (61527, Active Motif; 1:1000), histone H4 (ab177840, Abcam; 1:1000), GAPDH (2118S, Cell Signaling Technology; 1:5000), flag‐tag (F7425, Millipore; 1:5000), and V5‐tag (13202S, Cell Signaling Technology; 1:1000).

Techniques: CRISPR, Derivative Assay, Positive Control, Negative Control, Cryo-EM Sample Prep, Western Blot, Expressing, Competitive Binding Assay, Transduction, Co-Immunoprecipitation Assay, Binding Assay, Targeted Gene Expression

Journal: bioRxiv

Article Title: Assembly of mTORC3 involves binding of ETV7 to two separate sequences in the mTOR kinase domain

doi: 10.1101/2024.03.19.583970

Figure Lengend Snippet: A. Top drawing: schematic representation of the N- and C-terminally extended LBE fragment. The numbers indicate the number of amino acids in each of the LBE segments. Below is a FLAG immunoblot of an IP of the 120aa LBE fragment after overnight binding to ETV7 in vitro . Control (CTRL) shows the binding of human RUVBL2 to ETV7. B. Left panel, schematic representation of the different deletions of the N- and C-terminal extended LBE fragment (120 amino acids) and underneath the amino acid sequence of the LBE domain with the amino acids essential for ETV7 binding in red; Right, FLAG immunoblot of an ETV7 IP of the different LBE fragments after overnight binding to ETV7 in vitro . Lanes containing fragments that lost binding to ETV7 are boxed in red. C. FRB and LBE bind to ETV7 simultaneously. FLAG immunoblot of an ETV7 IP of LBE (120 amino acids) and Ndel-FRB fragments (ETV7: LBE: Ndel-FRB = 1:1:1, 1:1:5, 1:5:1) after overnight binding to ETV7 in vitro . D. Binding of Nde-FRB to ETV7 fragments containing the PNT domain and binding of LBE (120 amino acids) to ETV7 fragments containing the PNT and ETS domains in vitro .

Article Snippet: ETV7 protein (Cat # TP307742) and RUVBL2 (Cat # TP300933) were purchased from Origene.

Techniques: Western Blot, Binding Assay, In Vitro, Control, Sequencing

Journal: JCI Insight

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens

doi: 10.1172/jci.insight.172449

Figure Lengend Snippet: ( A ) Proteins that were immunoprecipitated by recombinant antibody 19-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. The proteins, RUVBL1 and RUVBL2, were identified by mass spectrometry. ( B ) MiaPaca2 cells were costained with 19-3 and an antibody specific for RUVBL2. ( C ) The binding of incremental concentrations of 19-3 to RUVBL2 was measured by ELISA. ( D ) Western blot analysis of recombinant antibody 15-7 detection of cytoplasmic proteins (Cyto) and mitochondria enriched proteins (Mito) is shown. ( E ) Immunofluorescence staining of MiaPaca2 cells by 15-7 and an antibody specific for HSPD1 is shown. ( F ) The binding of incremental concentrations of 15-7 to HSPD1 was measured by ELISA. ( G ) The serum IgG titers of normal individuals ( n = 61) and PDAC patients ( n = 59) to F-actin (sera diluted 1:100), to RUVBL2 (sera diluted 1:1,000), and to HSPD1 (sera diluted 1:1,000), respectively, were measured by ELISA. Background ELISA signal is indicated by dashed line. Individual data and mean are shown. The nonparametric t test was used for comparison. Each experiment was repeated at least twice. Scale bars in B and E are 50 μm.

Article Snippet: Other antibodies and dyes used were Phalloidin (Thermo Fisher Scientific, A12379), MYH10 (Atlas Antibodies, HPA047541), cytochalasin D (Thermo Fisher Scientific, PHZ1063), Actin (Cell Signaling Technology, 3700S), RUVBL2 (Atlas Antibodies, HPA067966), RUVBL1 (Atlas Antibodies, HPA019947), HSPD1 (Atlas Antibody, HPA050025), HSPA9 (Atlas Antibody, HPA000898), COX4I1 (Atlas antibody, HPA002485), KRT19 (Abcam, ab203445), and human IgG isotype controls (BioLegend, 403502 and 403702).

Techniques: Immunoprecipitation, Recombinant, SDS Page, Silver Staining, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Comparison

Journal: JCI Insight

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens

doi: 10.1172/jci.insight.172449

Figure Lengend Snippet: ( A ) Proteins that were immunoprecipitated by recombinant antibody 19-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. The proteins, RUVBL1 and RUVBL2, were identified by mass spectrometry. ( B ) MiaPaca2 cells were costained with 19-3 and an antibody specific for RUVBL2. ( C ) The binding of incremental concentrations of 19-3 to RUVBL2 was measured by ELISA. ( D ) Western blot analysis of recombinant antibody 15-7 detection of cytoplasmic proteins (Cyto) and mitochondria enriched proteins (Mito) is shown. ( E ) Immunofluorescence staining of MiaPaca2 cells by 15-7 and an antibody specific for HSPD1 is shown. ( F ) The binding of incremental concentrations of 15-7 to HSPD1 was measured by ELISA. ( G ) The serum IgG titers of normal individuals ( n = 61) and PDAC patients ( n = 59) to F-actin (sera diluted 1:100), to RUVBL2 (sera diluted 1:1,000), and to HSPD1 (sera diluted 1:1,000), respectively, were measured by ELISA. Background ELISA signal is indicated by dashed line. Individual data and mean are shown. The nonparametric t test was used for comparison. Each experiment was repeated at least twice. Scale bars in B and E are 50 μm.

Article Snippet: Recombinant proteins RUVBL2 (Creative Biomart, RUVBL2-30950), RUVBL1 (Novus, NBP1-50845), HSPD1 (Origene, TP760396), or BSA control were coated in PBS with 0.25 μg per well in a 96-well ELISA plate (Corning) overnight in 4°C.

Techniques: Immunoprecipitation, Recombinant, SDS Page, Silver Staining, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Comparison