Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy

doi: 10.1080/15548627.2024.2440846

Figure Lengend Snippet: BPLF1 does not have deUfmylase activity. (A) Representative immunoblots of an in vitro UFMylation reaction performed in the presence or absence of BPLF1 or UFSP2. UFMylation reactions were performed in the presence of purified components of conjugation cascade: 0.25 μM recombinant His-UBA5 (E1), 5 μM GST-UFC1 (E2), 1 μM His-UFL1/Strep-DDRGK1 (E3), 0.5 μM of H3/H4 complex (substrate), and 10 μM His-UFM1 in the absence (lane 1) or presence of 5 mM ATP (lanes 2–9). Where indicated increasing concentrations of recombinant BPLF1 (lanes 3–5; 0.3, 0.75, and 1.5 μM, respectively), His-BPLF1 C61A (CM, lane 6; 1.5 μM) or His-UFSP2 (lane 7–9; 0.03, 0.075, and 0.15 μM, respectively) were included in the reaction. The DUB activity of recombinant BPLF1 was confirmed by labeling 1.5 μM His-BPLF1 with 1 μM of the functional probe HA-Ubiquitin-vinyl-sulphone (Ub-Vs) that forms covalent adducts with the catalytic cys residue (lane 10–11). All reactions were incubated at 37°C for 90 min and analyzed in immunoblots using the indicated antibodies. UFMylated H3/H4, UFL1, and DDRGK1 were detected when the reaction was performed in the presence of ATP (lane 2). The addition of increasing amounts of BPLF1 or BPLF1 C61A had no effect (lanes 3–6), while efficient de-UFMylation was induced by minute amounts of UFSP2 (lanes 7–9). The experiment was repeated twice with comparable results. UFM1 dimers were detected by the UFM1 antibody as a strong band of approximately 20 kDa. (B) recombinant BPLF1 does not cleave a UFM1-GFP reporter. Increasing amounts of purified recombinant BPLF1 (0.3, 0.75, 1.5 µM) were mixed with the UFM1-GFP or Ub-GFP reporters (0.3 µM) in reaction buffer and incubated for 1 h at 37°C followed by immunoblot analysis. One representative experiment out of three is shown in the figure. (C) Representative immunoblots illustrating the failure of BPLF1 to cleave a UFM1-GFP reporter in cells. U2OS- UFSP2 -knockout cells were cotransfected with a UFM1-GFP fusion protein-expressing plasmids and plasmids expressing BPLF1 or the cellular deUfmylase UFSP2. The production of free GFP and UFM1 was monitored in immunoblots probed with specific antibodies. The reporter was cleaved only in cells expressing UFSP2. One representative experiment out of two is shown.

Article Snippet: pCMV3-UFSP2-FLAG , Sino Biological , HG17133-CF.

Techniques: Activity Assay, Western Blot, In Vitro, Purification, Conjugation Assay, Recombinant, Labeling, Functional Assay, Residue, Incubation, Knock-Out, Expressing

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy

doi: 10.1080/15548627.2024.2440846

Figure Lengend Snippet: BPLF1 does not affect the assembly and activity of the UFL1 ligase complex. (A) reciprocal co-immunoprecipitation assays illustrate the failure of BPLF1 to affect the interaction of UFL1 with DDRGK1 and CDK5RAP3 in untreated cells. Lysates of HEK293T transfected with FLAG-ev, -BPLF1 or -BPLF1 C61A were divided into equal aliquots, and co-immunoprecipitation was carried out with the indicated antibodies. Comparable levels of catalytic active and mutant BPLF1 were detected by the FLAG antibody in the UFL1, DDRGK1, and CDK5RAP3 immunoprecipitates, but their presence did not affect the co-immunoprecipitation of the ligase components. One representative experiment out of three is shown in the figure. (B) BPLF1 does not affect the assembly of the ligase in ANS-treated cells. HEK293T were transfected with FLAG-ev or -BPLF1, and one aliquot of the FLAG-BPLF1 transfected cells was treated with 50 ng/ml ANS for 30 min before harvesting. Immunoprecipitates of endogenous DDRGK1 were probed with the indicated antibodies. Immunoblots from one out of two independent experiments are shown. (C) BPLF1 does not affect the activity of the UFL1 ligase. The UFMylation of CYB5R3 was induced in HEK293- UFSP2 -knockout cells by transfection of ha-tagged UFL1 and DDRGK1 and MYC-tagged active UFM1 (UFM1ΔC2) with or without co-transfection of FLAG-BPLF1 or -UFSP2. Representative immunoblots from one of two independent experiments are shown.

Article Snippet: pCMV3-UFSP2-FLAG , Sino Biological , HG17133-CF.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Mutagenesis, Western Blot, Knock-Out, Cotransfection

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy

doi: 10.1080/15548627.2024.2440846

Figure Lengend Snippet: Reagents used in this paper.

Article Snippet: pCMV3-UFSP2-FLAG , Sino Biological , HG17133-CF.

Techniques: Recombinant, Diagnostic Assay, Transfection, Mutagenesis, Cloning, cDNA Synthesis, SYBR Green Assay, DC Protein Assay, Protein Purification, Knock-Out, Plasmid Preparation

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy

doi: 10.1080/15548627.2024.2440846

Figure Lengend Snippet: PCR and qPCR primers.

Article Snippet: pCMV3-UFSP2-FLAG , Sino Biological , HG17133-CF.

Techniques: FLAG-tag

Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmid coding for E1 Ubiquitin-Activating Enzyme 1 (UBA1) (pCMV3-UBA1-c-Myc) was purchased from SiNo Biological (Düsseldorfer, Eschborn, Germany).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Cotransfection

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A & B. Primary human fibroblasts were pre-labeled by DiI (red) and MDA-MB-231 (A) or BT549 cells (B) were pre-labeled by DiD (blue). Cells were cocultured for overnight before fixation for IF staining. Cadherin-11 protein was stained by the specific monoclonal primary antibody (clone 16A) followed by the secondary antibody staining (green). Purple arrows are pointing to the cadherin-11 AJs between cancer cells and fibroblasts. Confocal Z-stacks were scanned from the top of the cell to the bottom of the cell. All 2-D images shown were from 3-D Z-stacks maximum projections. C. The schematic to describe the cell invasion assay. D. Fibroblasts and MDA-MB-231 cells were pre-labeled as before. Fibroblasts alone (middle panels), MDA-MB-231 alone (right panels) or Fibroblasts and MDA-MB-231 (left panels) together in a 1:1 ratio were subjected to the cell invasion assay as described in (C). The whole cell population invasion displacements on the X-axis with a direction to the left were labeled between the yellow lines of 0 hr and 16 hr. The green fluorescence from the matrigel was omitted to clearly visualize the cells. Size bar, 100 μm. E, F & G. Cell invasion speed (Distance/time), invasion velocity (Displacement on the X-axis/time) and invasion persistence (Displacement/Distance) were quantitated. *, P < 0.05. H. Time-lapse zoom-in panels from Video 3. Green arrows are pointing at one cancer cell that was invading back-n-forth by attaching to and sliding on the cell bodies of fibroblasts. The red channel imaging offsets were elevated to visualize the long but thin invasive protrusions of fibroblasts. Size bar, 100 μm. I. Cropped and zoom-in panels from (H) to present the details of the invasive protrusions of fibroblasts (yellow arrow heads).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Staining, Invasion Assay, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were pre-labeled by DiI (red) and BT549 cells were pre-labeled by DiO (green). BT549 alone spheroid with negative control siRNA or CDH11 siRNA (top panels), and fibroblasts alone spheroid with negative control siRNA or CDH11 siRNA (bottom panels) were subjected to the 3D spheroid cell invasion assay. B. Quantitation for images as in (A). Experiments were repeated 6 times (n=6). *, P < 0.05. C. BT549 and fibroblasts coculture (1:1) spheroid with negative control siRNA or CDH11 siRNA were subjected to the 3D spheroid cell invasion assay. D, E & F. Quantitation for images as in (C). Experiments were repeated 6 times (n=6). *, P < 0.05. Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Negative Control, Invasion Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 (Cadherin-11), ESR1(Estrogen Receptor 1), PGR (Progesterone Receptor) & ERBB2 (Receptor tyrosine-protein kinase erbB-2) expression data in breast cancer cell lines from CCLE (Cancer Cell Line Encyclopedia, The Broad Institute of MIT & Harvard) were plotted into a heat map. B. Kaplan-Meier analysis for breast cancer patients stratified by CDH11 expression for 1075 patients from The Human Protein Atlas database. C-E. Kaplan-Meier plots of overall survival (C, n=626), relapse-free survival (D, n=1764) and distant metastasis free survival (E, n=664) of breast cancer patients in relation to CDH11 expression according to The KM-plotter database. F. Kaplan-Meier plots of distant metastasis free survival (n=68) of ER negative breast cancer patients in relation to CDH11 expression according to The KM-plotter database.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were transduced by GIPZ lentiviral CDH11 shRNA or non-silencing shRNA with a GFP reporter. Stable transductant cells were sorted out by FACS based on the GFP signal. Silencing of CDH11 in these cells was then quantified by RT-qPCR. B. Effect of CDH11 silencing in human fibroblasts on cancer cell growth in the cancer with fibroblast co-implantation xenograft mouse model. 1 × 10 6 of MDA-MB-231-luc cells mixed with 1 × 10 6 of stable non-silencing (blue) or CHD11 silencing (red) primary human fibroblasts were co-implanted into the left fourth mammary fat pad of NOD/SCID mice. The bioluminescence of the MDA-MB-231-Luc cells were measured every 2 weeks. C. Representative in vivo bioluminescence images from (B) at 14 weeks after cancer with fibroblast co-implantation. D. Comparison of tumor volume in mice co-implanted with MDA-MB-231-luc cells and non-silencing or CDH11 silencing primary human fibroblasts. Data were presented as mean ± SD (n=8). P values were determined by two-tailed Student’s t tests (NS, not significant; *, 0.01 < p < 0.05).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: shRNA, Quantitative RT-PCR, In Vivo, Two Tailed Test

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 stable overexpression in 4T1 cells (4T1-CDH11) was quantified by RT-qPCR against CDH11 expression levels in 4T1 wildtype cells (4T1-WT). B. 4T1-WT cells or 4T1-CDH11 cells were pre-labeled by DiD (red). NIH3T3 mouse fibroblasts were pre-labeled by DiO (green). 4T1 alone spheroids (top panels), or 4T1 and NIH3T3 coculture (1:1) spheroids (bottom panels) were subjected to the 3D spheroid cell invasion assay as in . Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections. C. Quantitation for images as in (B). Experiments were repeated 5 times (n=5). NS, not significant; *, P < 0.05. D. Comparison of tumor volume in BALB/c mice implanted with 4T1-WT cells or 4T1-CDH11 cells. 1 × 10 6 of 4T1 cells were implanted into the left fourth mammary fat pad in BALB/c mice. Data were presented as mean ± SD (n=8). E. Comparison of 4T1-WT cell and 4T1-CDH11 cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. F. Kaplan-Meier survival curve of BALB/c mice implanted with either 4T1-WT cells or 4T1-CDH11 cells as in (D), n=8. G. micro-MRI imaging of tumor-bearing BALB/c mice from (D) on the 21 st day after implantation of 4T1-WT cells or 4T1-CDH11 cells. Multiple distal metastatic sites in the dorsal neck region lymph nodes (denoted by small arrows) were detected in mice with 4T1-CDH11 tumors. Large arrowheads denote the original tumors at the left fourth mammary fat pad. micro-MRI image Z-stacks were scanned from the dorsal side to the ventral side of the mice. Single plane image section across the dorsal neck region lymph nodes from two representative mice from each group is shown. No distal metastasis was detected in any mice with 4T1-WT tumors in all micro-MRI image Z-stacks.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Labeling, Invasion Assay, Quantitation Assay, In Vitro, Micro-MRI, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: 1 × 10 6 of 4T1 mouse triple negative breast cancer cells expressing firefly luciferase with or without CDH11 overexpression were implanted into the left fourth mammary fat pad of immunocompetent BALB/c mice (A-C) or NOD/SCID mice (E-G). A. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in BALB/c mice. B. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in BALB/c mice. Data were presented as mean ± SD (n=7). C. Representative in vivo bioluminescence images from (B). D. Comparison of 4T1-WT-luc cell and 4T1-CDH11-luc cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. E. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in NOD/SCID mice. F. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in NOD/SCID mice. Data were presented as mean ± SD (n=5). G. Representative in vivo bioluminescence images from (F).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing, Luciferase, Over Expression, In Vivo, In Vitro