Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: The depletion of USP39 suppresses proliferation and induces cell death in OPM2 myeloma cell line

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 is overexpressed in MM patients compared to healthy donors and its high expression is correlated with shorter survival. A Kaplan–Meier of overall survival in patients with MM with high (red line) or low (black line) USP39 mRNA expression (P = 0.038) (GEO dataset GSE9782). B USP39 mRNA expression in normal donor, MGUS, Smoldering and MM patients (GEO dataset GSE6477). C Left, Representative USP39 staining of bone marrow samples from healthy individuals. Right, Representative USP39 staining of bone marrow samples from MM patients. “NR” denotes a non-relevant antibody. D Representative USP39 immunostaining of bone marrow samples from MM patients. Staining intensity was interpreted by a pathologist using visual scoring: "– “ undetectable, “ + ” denotes low intensities, “ + + ” denotes medium intensities, and “ + + + ” denotes high intensities. E Table representing USP39 staining of 12 bone marrows from MM patients and 6 bone marrows from healthy individuals. The percentage of USP39 positive cells was determined by ImageJ quantification and confirmed by pathologist visual scoring. Staining intensity were interpreted by pathologist visual scoring: "– “ denotes undetectable, “ + ” denotes low intensities, “ + + ” denotes medium intensities, and “ + + + ” denotes high intensities

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Expressing, Staining, Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Depletion Suppresses Cell Proliferation, Induces Apoptosis, and decreases Clonogenicity in OPM2 and KMM1 Multiple Myeloma Cells. A OPM2 cells were transfected with either control or two different single USP39 siRNA (siUSP39 #1 and siUSP39 #2) for 96 h. Then, lysates from these cells were subjected to immunoblots using GAPDH and USP39 antibodies (upper part). In parallel, the percentage of cell death was measured by flow cytometry after IP staining (left lower part) and cell metabolism was assessed by XTT assay (right lower part). B KMM1 were treated as described for OPM2 cells and subjected to the same analysis. C OPM2 cells were transfected with either control or single USP39 siRNAs for 96 h or stimulated with BTZ for 48 h. Lysates from these cells were subjected to immunoblots using GAPDH and USP39 antibodies (upper left). In parallel, the clonogenic capacity of the cells was measured after 10 days within a semi-solid medium. The quantification of the clonogenic assay is reported in the upper right part of the figure. Representative pictures were shown in the lower part. D KMM1 were treated as described for OPM2 cells and subjected to the same analysis. E KMM1 cells were either transfected with Control or USP39 siRNAs for 24 h. Then cells were transfected with Myc-Tag or Myc-USP39 vectors. After 72 h, lysates from these cells were subjected to immunoblots using GAPDH, myc-Tag or USP39 antibodies (left part). After 96 h of transfections, cell metabolism was measured in each condition (right part)

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Transfection, Control, Western Blot, Flow Cytometry, Staining, XTT Assay, Clonogenic Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: Inhibition of USP39 Triggers G2/M Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells. A OPM2 cells were transfected with control or USP39 siRNAs for 72 h, 96 h or 120 h. In parallel, cells were stimulated with nocodazole (1 µg/ml) for 24 h to block the cells in G2/M phase. Cell cycle distribution was examined by flow cytometry, and percentage of cells in each phase is indicated (top left and right). A representative flow cytometry profile of cells transfected with control (blue area) or USP39 siRNAs (red area) for 96 h (bottom left). B In parallel, OPM2 cells were transfected with either control or single USP39 siRNAs for 72 h. Then, lysates from these cells were subjected to immunoblots using GAPDH, USP39, CDK4 and CyclinB1 antibodies. C OPM2 cells were transfected with either control or single USP39 siRNAs for 96 h or stimulated with BTZ for 48 h. Then, cells were stained by Annexin and PI and analyzed by flow cytometry. % of apoptotic cells (annexin V + /DAPI-) and dead cells (annexin V + /DAPI +) are represented in grey and black respectively. D Lysates from these cells were subjected to immunoblots using USP39, PARP, cleaved caspase 3 and GAPDH antibodies as a loading control. E , F OPM2 cells were transfected with either control or single USP39 siRNAs for 72 h and 96 h, or stimulated with BTZ for 48 h. Then, cells lysates were subjected to caspase 3 (E) and caspase 9 (F) assays

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Inhibition, Transfection, Control, Blocking Assay, Flow Cytometry, Western Blot, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Inhibition Overcomes Bortezomib Resistance in MM Cells. A U266 cells and its BTZ-resistant counterpart U266R were stimulated with increased concentrations of BTZ (1, 3, 10, 30 and 100 ng/ml) for 24 h, and cell metabolism was measured by XTT assay. B U266 and U266R cells were transfected with either control or three different single USP39 siRNAs (#1, #2 and #3) for 72 h. USP39 silencing was confirmed by immunoblots using USP39 and GAPDH antibodies (top). In parallel, cell metabolism was measured by XTT assay (bottom). C U266 and U266R cells were transfected with either control or two different single USP39 siRNAs (#1 and #2) for 72 h. Then both cells were stimulated with increased concentrations of BTZ (1, 3, 10, 30 and 100 ng/ml) for 24 h and cell metabolism was measured by XTT assay

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Inhibition, XTT Assay, Transfection, Control, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Stabilizes and Deubiquitinates ZEB1 Protein in Multiple Myeloma Cells . A OPM2 cells were transfected with either control or USP39 siRNAs for 48 h, 72 h or 96 h. Lysates were subjected to immunoblots using USP39, ZEB1, SP1, CHK2, STAT1 and GAPDH antibodies. B USP39 was transiently silenced or overexpressed in OPM2 and KMM1 cells respectively. Then, immunoblots were performed using USP39, ZEB1 and GAPDH antibodies (left) and protein quantifications were determined (right). C KMM1 cells were transfected with plasmids encoding either the Myc-tag or the USP39-Myc-tag proteins. After 48 h, cells were stimulated with cycloheximide (CHX) at 10 µM for 24 h. Lysates were subjected to immunoblots using USP39, ZEB1, and GAPDH antibodies and protein quantification was determined. D KMM1 cells were transfected with either control or USP39 siRNAs. After 72 h, cells were stimulated with cycloheximide (CHX) at 10 µM for 24 h. Lysates were subjected to immunoblots using USP39, ZEB1, and GAPDH antibodies and protein quantification was determined. E OPM2 cells were transfected with either control or USP39 siRNAs for 72 h. Then cells were stimulated for 2 h, 4 h or 8 h with the proteasome inhibitor MG132 at 1 µM. Lysates were subjected to immunoblots using USP39, ZEB1 and GAPDH antibodies. Protein quantification was determined. F Lysates from OPM2 cells were subjected to co-immunoprecipitation experiments using either non relevant (NR), USP39 or ZEB1 antibodies. Immunoblots was performed to visualize complexes using USP39 and ZEB1 antibodies. G KMM1 cells were transfected for 48 h with HA-ub plasmid in the presence or in the absence of Myc-USP39 plasmid. Then cells were treated with MG132 at 1 µM for 8 h and lysates were subjected to immunoprecipitation using non relevant IgG or ZEB1 antibodies. Inputs were immunoblotted with HA and Myc antibodies to visualize poly-HA-Ub and USP39 respectively. IPs products were immunoblotted with HA and ZEB1 antibodies to visualize Ub-ZEB1 complex and immunoprecipitated ZEB1. The graph represents Ub-ZEB1 quantification. H The complementary deubiquitination experiment was performed in presence or absence of USP39 siRNA (72 h)

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Promotes In Vitro Transmigration of MM Cells. A OPM2 cells were transfected with control, USP39 or ZEB1 siRNAs for 48 h. Then, immunoblots were performed using USP39, ZEB1, β-Catenin, N-Cadherin, Vimentin and GAPDH antibodies (left) and protein quantifications were determined (right). In parallel, the metabolic activity (lower left) and the migration of the cells (lower right) were measured under the same conditions. B KMM1 cells were either transfected with Control or USP39 siRNAs for 24 h. Then cells were transfected with pcDNA3-Flag or pcDNA3-Flag-USP39 vectors. After 72 h, lysates from these cells were subjected to immunoblots using USP39, Flag-Tag or GAPDH, antibodies (left part). After 96 h of transfections, the migration capacity of cells was measured by Boyden chamber assays (right part). C and D ) U266 ( C ) and OPM2 ( D ) cells were stably transduced with lentiviral particles encoding GFP or GFP-USP39 and were subjected to immunoblots using USP39, ZEB1 or GAPDH antibodies. E , F U266 ( E ) and OPM2 ( F ) cells were stably transfected with lentiviral particles encoding Myc or Myc-USP39 and were subjected to immunoblots using USP39, ZEB1 or GAPDH antibodies. In parallel, the migration capacity of corresponding cells was measured by Boyden chamber assays

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: In Vitro, Transmigration Assay, Transfection, Control, Western Blot, Activity Assay, Migration, FLAG-tag, Stable Transfection, Transduction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Enhances Metastasis in Zebrafish: Implications for MM Progression. A-D Zebrafish embryos ( N = 36) were injected with U266 cells stably infected with lentiviral particles encoding either Myc-tag or Myc-USP39 proteins (labeled with red DiD) into the perivitelline space. Zebrafish embryos were monitored on Day 0 and Day 2 for tumor metastases using a fluorescent microscope. A (Left) Representative images of local and distant metastases are shown. (Right) Magnification of images representing distant metastases. Arrows indicate metastases. Quantification of the area of local metastases at Day 0 ( B ) and Day 2 ( C ) of Myc-tag and Myc-USP39 embryos. D Quantification of the number of distant metastases at Day 2 of Myc-tag and Myc-USP39 embryos

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Injection, Stable Transfection, Infection, Labeling, Microscopy

Journal: Cell reports

Article Title: FOXR2 Interacts with MYC to Promote Its Transcriptional Activities and Tumorigenesis

doi: 10.1016/j.celrep.2016.06.004

Figure Lengend Snippet: (A) Prediction of transcription factors' involvement in tumorigenesis is based on their HCIPs identified by TAP-MS analysis. The cancer correlations were generated by searching the HCIP datasets of each transcription factors in the knowledge base to estimate the significance of these correlations. Transcription factor interactomes were searched for their alteration (numbers and rates) in multiple TCGA databases using their HCIP sets. X axis indicates the relative average expression alteration of indicated TF HCIP dataset in multiple TCGA databases. Y axis indicates cancer correlation for each transcription factor, estimated on the basis of their HCIPs identified in chromatin fractions. The size of each dot indicates the relative average mutation rate of TF HCIP dataset in multiple TCGA databases. (B) Top HCIPs of FOXR1 and FOXR2 in HEK293T cells are listed together with their NSAF values. Baits are highlighted in blue; MYC and MAX are highlighted in orange. (C) Schematic representation of FOXR1 and FOXR2 interactomes with top-ranked interacting proteins. The interaction networks were visualized using unweighted force-directed distributions. Purple lines indicate interactions defined by the literature. Grey lines indicate newly identified interactions on the basis of the results of our proteomic study. Orange dots indicate MYC-MAX complex members reported in the literature. (D) FOXR2 expression in HEK293T, MCF10A and MDA-MB-468 cells were evaluated using whole proteome profiling and WB analysis with antibodies against endogenous FOXR2. (E) TAP-MS was performed with MDA-MB-468 cells that stably expressed SFB-tagged FOXR2, MYC, or MAX. Top-ranked interacting proteins are listed together with their NSAF values. Baits are highlighted in blue; prey FOXR2, MYC, and MAX are highlighted in orange. (B and E) All the data listed here are statistical significant.

Article Snippet: Lysates were subjected to SDS-PAGE, followed by immunoblotting with antibodies against various proteins, including MYC, FOXR2, GFP, GAPDH, MAD1 (Santa Cruz Biotechnology), cleaved Caspase-3, MYC, MAX (Cell Signaling), FOXR2 (Abcam), β-actin, FLAG (Sigma), and histone H3 (Upstate).

Techniques: Generated, Expressing, Mutagenesis, Stable Transfection

Journal: Cell reports

Article Title: FOXR2 Interacts with MYC to Promote Its Transcriptional Activities and Tumorigenesis

doi: 10.1016/j.celrep.2016.06.004

Figure Lengend Snippet: (A) Upper panel: Co-IP of endogenous MAX or MYC with FOXR2 was performed with IgG or anti-FOXR2 antibody using soluble and chromatin fractions prepared from MDA-MB-468 cells. 5% of the corresponding cell lysate used in the IP was included as input control. Immunoblotting was conducted using the indicated antibodies. Lower panel: Co-IP of endogenous MAX or MYC with FOXR2 was performed with anti-FOXR2 antibody or pre-immune serum from the same mouse, using chromatin fractions prepared from MDA-MB-468 and MDA-MB-468/sh-FOXR2 cells. (B) Immunodepletion of FOXR2 and MYC performed using extracts prepared from MDA-MB-468 cells. MDA-MB-468 cell lysates were immunoprecipitated with FOXR2 or MYC antibodies three times. FOXR2, MYC, MAX, and β-actin levels were measured after each round. (C) Cell lysates of MDA-MB-468 and MDA-MB-468/sh-FOXR2 cells were immunoprecipitated with MYC or MAX antibodies and immunoblotted with the indicated antibodies. (D) HEK293T cells were transfected with constructs encoding the indicated FOX proteins. Co-IP experiments were performed using S-protein beads and blotted with antibodies recognizing the Flag-epitope tag or endogenous MYC. 5% of the corresponding cell lysate used in the IP was included as input control. Only FOXR1 and FOXR2 were able to pull down endogenous MYC. (E) Schematic diagram of MYC mutants is shown. The bHLH domain is depicted as dark grey. The mutants were: D1: (Δ1-120); D2: (Δ120-240); D3: (Δ240-350); D4: (Δ350-439). SFB-tagged wild-type and mutants of MYC were subjected to co-precipitation experiments with endogenous FOXR2 in MDA-MB-468 cells. 5% of the corresponding cell lysate used in the IP was included as input control. (F, G) Schematic diagram of FOXR2 mutants is shown. The FOX domain appears as dark grey. The mutants were: ΔN: (Δ1-113); ΔI: (Δ113-192); ΔC: (Δ192-311); D1: (Δ1-20); D2: (Δ20-40); D3: (Δ40-60); D4: (Δ60-80); D5: (Δ80-100); D6: (Δ100-113). SFB-tagged wild-type and mutants of FOXR2 were subjected to co-precipitation experiments with endogenous MYC in HEK293T cells. 5% of the corresponding cell lysate used in the IP was included as input control.

Article Snippet: Lysates were subjected to SDS-PAGE, followed by immunoblotting with antibodies against various proteins, including MYC, FOXR2, GFP, GAPDH, MAD1 (Santa Cruz Biotechnology), cleaved Caspase-3, MYC, MAX (Cell Signaling), FOXR2 (Abcam), β-actin, FLAG (Sigma), and histone H3 (Upstate).

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Immunodepletion, Immunoprecipitation, Transfection, Construct, FLAG-tag

Journal: Cell reports

Article Title: FOXR2 Interacts with MYC to Promote Its Transcriptional Activities and Tumorigenesis

doi: 10.1016/j.celrep.2016.06.004

Figure Lengend Snippet: (A) Chromatin IP-sequencing (ChIP-seq) assay was performed in MDA-MB-468 cells using endogenous antibodies against FOXR2, MYC or MAX. Overlap between FOXR2 and MAX or MYC target genes were evaluated. (B) The global analyses of relative peak positions of FOXR2 and MYC or MAX were shown. (C) A chromatin IP (ChIP) assay was performed in MDA-MB-468 cells using FOXR2 antibody or control IgG. The recovery of MYC downstream gene promoter regions was examined by real-time PCR. All the promoters except the control p15-distal promoter have significantly enriched in the FOXR2 (P < 0.001) but not IgG immunoprecipitants. (D) A ChIP-reChIP assay was performed in MDA-MB-468 cells overexpressing SFB-tagged FOXR2 using streptavidin-beads, eluted with biotin and re-IPed with MYC-antibody or control IgG. The recovery of MYC downstream gene promoter regions was examined by real-time PCR. All the promoters except the control p15-distal promoter have significantly enriched in the MYC (P < 0.001) but not IgG immunoprecipitants. (E) MYC target gene expression profiles were evaluated by RT PCR in MCF10A, MCF10A-FOXR1, -R2, -R1 (ΔN), and -R2 (ΔN) cells. mRNA levels were determined by real-time RT-PCR and normalized with GAPDH. CCNA, CCND1 (P < 0.01) and COL1A1 (P < 0.05) expression levels have been significantly changed in MCF10A-FOXR1/R2 cells comparing with MCF10A cells. (F) Knocking down FOXR2 in MDA-MB-468 cells and reconstitution with wild-type or D5 mutant of FOXR2 were confirmed by immunoblotting, as indicated. (G) MYC target gene expression profiles were evaluated by RT PCR in MDA-MB-468, MDA-MB-468-shFOXR2, MDA-MB-468-shFOXR2+SFB-FOXR2 and MDA-MB-468-shFOXR2+SFB-FOXR2(D5) cells. mRNA levels were determined by real-time RT-PCR and normalized with GAPDH. CCNA, CCND1 and COL1A1 expression levels have been significantly changed in MDA-MB-468-shFOXR2 and MDA-MB-468-shFOXR2+SFB-FOXR2(D5) cells (P < 0.01), comparing with MDAMB-468 cells. (H) Growth curve of MCF10A derivative cells used in (C). MCF10A-FOXR1/R2 cells have significantly more cell numbers after 3 days (P < 0.01), comparing with MCF10A cells. Data are averages (± SD) of three independent experiments. (I) Growth curves of MDA-MB-468 parental and derivative cells used in (E) are shown. MDA-MB-468-shFOXR2 and MDA-MB-468-shFOXR2+SFB-FOXR2(D5) cells have significantly less cell numbers after 3 days (P < 0.01), comparing with MDA-MB-468 cells. (J, K) Soft agar colony formation of MCF10A cells that stably expressed indicated proteins was assessed and is presented. Colony numbers: MCF10A-FOXR1: 12.4 ± 2.2; MCF10A-FOXR1(ΔN): 1.4 ± 1.3; MCF10A-FOXR2: 14.2 ± 2.4; MCF10A-FOXR2(ΔN): 0.8 ± 0.8; MCF10A-FOXR2(D1): 9.7 ± 1.8; MCF10A-FOXR2(D5): 2.8 ± 1.9. The P < 0.001 between MCF10A-FOXR1 and MCF10A-FOXR1(ΔN); MCF10A-FOXR2 and MCF10A-FOXR2(ΔN); MCF10A-FOXR2(D1) and MCF10AFOXR1(D5).

Article Snippet: Lysates were subjected to SDS-PAGE, followed by immunoblotting with antibodies against various proteins, including MYC, FOXR2, GFP, GAPDH, MAD1 (Santa Cruz Biotechnology), cleaved Caspase-3, MYC, MAX (Cell Signaling), FOXR2 (Abcam), β-actin, FLAG (Sigma), and histone H3 (Upstate).

Techniques: Chromatin Immunoprecipitation, Sequencing, ChIP-sequencing, Control, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Mutagenesis, Western Blot, Stable Transfection

Journal: Cell reports

Article Title: FOXR2 Interacts with MYC to Promote Its Transcriptional Activities and Tumorigenesis

doi: 10.1016/j.celrep.2016.06.004

Figure Lengend Snippet: (A) MCF10A cells were infected in six-well plates with retroviruses encoding RAS, MYC, FOXR2, or any two together. Cells were counted after 96 hours of infection. Normalized cell numbers are presented. Data are averages (± SD) of three independent experiments. Cell lysates of each cell lines were immunoblotted with the indicated antibodies. (B) Soft agar colony formation of MCF10A cells that stably expressed the indicated proteins was assessed and is presented. (C) Ki-67 and BrdU staining of MDA-MB-468 and MDA-MB-468/sh-FOXR2 cells was performed as indicated. The percentages of positive cells are summarized on the right. **P < 0.01. (D) Xenograft tumor growth studies. 5 × 106 MDA-MB-468 and MDA-MB-468/sh-FOXR2 cells were resuspended in 100 μL of Matrigel diluted with PBS at 1:1 ratio and injected subcutaneously into left and right flanks of 10 anesthetized 6- to 8-week-old female BALB/c nude mice respectively. 5 × 106 MDA-MB-468/sh-FOXR2+SFB-FOXR2 and MDAMB-468/sh-FOXR2+SFB-FOXR2(D5) cells were resuspended in 100 μL of Matrigel diluted with PBS at 1:1 ratio and injected subcutaneously into left and right flanks of 10 anesthetized 6- to 8-week-old female BALB/c nude mice respectively. Starting from the day 0, the tumor weight and size were measured bi-weekly. Mice were euthanized after 8 weeks of injection. The tumors were excised, photographed, and weighed. *** P < 0.001. (E) Immunoblotting of FOXR2 in normal and breast cancer cell lines, with HEK293T cells as the negative control. (F) Immunoblotting of FOXR2 in normal and lung cancer cell lines. (F) Immunoblotting of FOXR2 in normal and liver cancer cell lines.

Article Snippet: Lysates were subjected to SDS-PAGE, followed by immunoblotting with antibodies against various proteins, including MYC, FOXR2, GFP, GAPDH, MAD1 (Santa Cruz Biotechnology), cleaved Caspase-3, MYC, MAX (Cell Signaling), FOXR2 (Abcam), β-actin, FLAG (Sigma), and histone H3 (Upstate).

Techniques: Infection, Stable Transfection, BrdU Staining, Injection, Western Blot, Negative Control

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 1 Nonselective and ADAM10-selective metalloprotease inhibitors increase the amount of full-length hGPR37 at the cell surface. A, Schematic model of hGPR37 with the metalloprotease cleavage site at Glu167↓Gln16816 and with three N-glycosylation sites16 shown as blue glycan structures. The added Myc and FLAG epitope tags are also indicated. B, HEK293i cells stably transfected with Myc-hGPR37-FLAG were induced to express the receptor for 24 hours and treated for the last 23 hours with the indicated metalloprotease inhibitors (20 µM marimastat, 2 µM GI254023X) or vehicle. Cells were fixed, permeabilized, and stained with the indicated antibodies followed by Alexa-Fluor-488- and -568- conjugated secondary antibodies. The nuclei were stained with TO-PRO-3 iodine. The cellular localization of cMyc antibody-labeled full-length receptors was analyzed by confocal microscopy. Scale bars: 10 µm. C, Induced HEK293i cells were treated with the indicated concentrations of GI254023X for 23 hours and analyzed by flow cytometry after labeling cell surface receptors with cMyc antibody and the phycoerythrin- conjugated secondary antibody. The results represent five independent experiments performed with triplicate samples. The fluorescence intensity values were normalized to the mean value obtained from cells treated with vehicle only. The results were analyzed before normalization using repeated measures one-way ANOVA followed by Dunnett's multiple comparison test. ***P < .001; **P < .01. Ab, antibody

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Glycoproteomics, FLAG-tag, Stable Transfection, Transfection, Staining, Labeling, Confocal Microscopy, Flow Cytometry, Fluorescence, Comparison

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 2 ADAM10 selective inhibitor GI254023X inhibits N-terminal cleavage of human and mouse GPR37. Stably transfected HEK293i cells were induced to express Myc-hGPR37-FLAG (A, C, G, H) or, alternatively, mGPR37-FLAG and Myc-hGPR37-FLAG were transiently expressed in either SH-SY5Y (E) or HEK293 (F) cells for 24 hours. The cells were treated with the indicated concentrations of protease inhibitors or vehicle for 20-23 hours. The shed N-terminal receptor fragment from the conditioned culture medium (A) and membrane-bound receptors from cellular lysates (C, E-H) were immunoprecipitated and analyzed by SDS-PAGE and Western blotting. The outlined area in panel E is shown with a longer exposure time. The identified hGPR37 species are depicted in panel B. The 67-kDa precursor and the 96-kDa full-length mature receptor are indicated with closed and open circles, respectively. The shed 32- 35-kDa N-terminal receptor fragment is indicated with a closed triangle and the 53- and 34-kDa cleaved C-terminal fragments with open and closed squares, respectively. The 70-kDa receptor species are indicated with an open triangle. Arrows in panel C indicate receptor oligomers and higher molecular mass aggregates. Panel D shows relative changes in the intensity of the 96-kDa full-length and 53-kDa cleaved receptors seen in panel C (left and right panels, respectively). The values were normalized to the 67-kDa precursor and are shown as means ± SD from four to nine independent experiments. The repeated-measures one-way ANOVA followed by Dunnett's multiple comparison test was used for the statistical comparison. **P < .01; *P < .05; ns, nonsignificant. Ab, antibody, decCMK, Decanoyl-RVKR-CMK; Hexa-D-Arg, hexa-D-arginine; IP, immunoprecipitation; WB, Western blotting

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Stable Transfection, Transfection, Membrane, Immunoprecipitation, SDS Page, Western Blot, Comparison

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 3 ADAM10 downregulation in HEK293 cells inhibits hGPR37 ectodomain cleavage and shedding. Inducible HEK293i cells were treated with siRNA pools targeting ADAM10 or ADAM17 or with the control siRNA pool for 72 hours and were induced to express hGPR37 for 8 hours at the end of the siRNA transfection. A, Receptors from solubilized cellular membranes were analyzed by SDS-PAGE and Western blotting. The knock-down of ADAM10 and ADAM17 was confirmed with the corresponding selective antibodies. The translocon-associated protein α-subunit (TRAPα) was used as a loading control. B, N-terminal receptor fragments in the cell culture medium were immunoprecipitated and analyzed by Western blotting with cMyc antibody. D, Cell surface cMyc antibody labeled receptors from five independent experiments were analyzed in triplicate by flow cytometry. Relative changes in the 96-kDa full-length and 53-kDa cleaved receptors seen in panel A are shown in panel C as means ± SD from 4-5 independent experiments. The values were normalized to the loading control before statistical analysis. The data were analyzed by repeated measures one-way ANOVA and Dunnett's multiple comparison test. *P < .05; ns, nonsignificant. The abbreviations and symbols describing GPR37 species are explained in the legend for Figure 2. i, immature ADAM17; m, mature ADAM10/17

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Control, Transfection, SDS Page, Western Blot, Knockdown, Cell Culture, Immunoprecipitation, Labeling, Flow Cytometry, Comparison

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 4 N-terminal cleavage of GPR37 is impaired in MEF and HEK293T cells lacking ADAM10 and is induced in vitro with rADAM10. The Myc- and FLAG-tagged hGPR37 was transiently expressed for 24 hours in WT and ADAM10KO MEF cells (A) or in WT, ADAM10KO, and ADAM17KO HEK293T cells that were treated or not with marimastat for 20 hours (B). For C-D, WT and ADAM10KO HEK293T cells were transiently transfected with Myc-hGPR37-FLAG together with the HA-tagged WT hADAM10 or its inactive E384A mutant (1:1 DNA ratio) for 48 hours. For E, mGPR37-FLAG was transiently expressed for 24 hours in WT and ADAM10KO HEK293T cells treated or not with marimastat for 20 hours, and for F, hGPR37 was expressed for 24 hours in stably transfected HEK293i cells treated with decCMK (10 µM) and GI254023X (5 µM) for 20 hours. Receptors and ADAM10 were immunoprecipitated from cellular lysates with FLAG and HA-antibodies, respectively, before analysis by Western blotting. For F, receptors were treated before elution from the FLAG antibody resin with rADAM10 as indicated. The additional GPR37 C-terminal fragment seen in HEK293T cells is indicated with an arrowhead (B, E). The other symbols and abbreviations are explained in the legends for Figures 2 and 3

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: In Vitro, Transfection, Mutagenesis, Stable Transfection, Immunoprecipitation, Western Blot

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 5 GPR37 is cleaved in its N-terminal domain by a non-metalloprotease. A, B and D, HEK293i cells were induced to express hGPR37 for 6 hours (A, B) or 16 hours (D), labeled with [35S]methionine/cysteine for 30 minutes and chased for various time periods before cell surface proteins were biotinylated (A) or not (B, D) with sulfo-NHS-biotin. GI254023X, decCMK, or vehicle were added to the medium during depletion, labeling, and chase. Receptors were subjected to two-step immunoprecipitation with FLAG antibody (A, upper panel, B, D) or were first purified with streptavidin agarose and then with FLAG antibody (A, lower panel) before analysis by SDS-PAGE and fluorography. For panel B, immunoprecipitated receptors from 120-minute chase samples were deglycosylated with Endo H (50 mU/mL) or PNGase F (20 U/mL) before SDS- PAGE. C, HEK293i cells were induced for 24 hours and treated with the indicated protease inhibitors or vehicle for 20 hours. Immunoprecipitated receptors were analyzed by SDS-PAGE and Western blotting. Note that GI254023X inhibits receptor cleavage more efficiently at 5 µM (B) than at 3 µM (A). PA, phosphoramidon. Other abbreviations and symbols are as described in the legends for Figures 2 and 3

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Labeling, Immunoprecipitation, Purification, SDS Page, Western Blot

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 6 Conservation of GPR37 N-terminal tail. WebLogo40 was used to generate a graphical representation of conservation between fourteen placental mammalian species (see Table S1). The generated sequence logo corresponding to residues 27-178 of hGPR37 (shown as a gray bar below the schematic model of the N-terminal domain) displays the conservation of each residue, indicated by the overall height of the letter. The relative height of each letter in the stack indicates the frequency of the corresponding amino acid at the site. The positively charged amino acids Arg and Lys are shown in red. The consensus sequence for N-glycosylation and putative PC cleavage sites are indicated with blue and yellow bars, respectively, below the sequence. The metalloprotease cleavage site (Glu167↓Gln168) identified for hGPR3716 is indicated with the red scissors. The corresponding sites for hGPR37 are also shown in the schematic model

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Generated, Sequencing, Residue, Glycoproteomics

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 7 GPR37 is cleaved at the conserved Arg54↓Asp55 site in the N-terminus by furin. HEK293 (A-E) and LoVo (F) cells were transiently transfected with the indicated Myc-hGPR37-FLAG constructs (A-D, F), or alternatively, with the NanoLuc- and HA-tagged hGPR37 (E) for 16-24 hours and treated or not with GI254023X (5 µM), decCMK (20 µM) or marimastat (20 µM) for 20 (A, B, F), 19 (C) or 12 (D, E) h. Immunoprecipitated receptors from cellular lysates (A, B, F) or aliquots of the concentrated conditioned culture medium without immunoprecipitation (D, E) were analyzed by SDS-PAGE and Western blotting. For panel C, cell surface cMyc antibody labeled receptors were analyzed by flow cytometry. Triplicate or quadruplicate samples from four independent experiments were analyzed by repeated-measures two- way ANOVA and Tukey's multiple comparison test before normalization to the respective controls without the inhibitor-treatment. *P < .05; ns, nonsignificant. Abbreviations and symbols are as in the legend for Figure 2

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Transfection, Construct, Immunoprecipitation, SDS Page, Western Blot, Labeling, Flow Cytometry, Comparison

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Plasmid Preparation, FLAG-tag, Variant Assay, Mutagenesis

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Cell Culture, Plasmid Preparation, Produced, Transfection, Bradford Assay, Detection Assay, Western Blot, Marker, Membrane

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Transduction, Plasmid Preparation, Produced, Cell Culture, Software

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Transduction, Plasmid Preparation, Produced, Cell Culture, Chromatography, Lysis, Protease Inhibitor, Magnetic Beads, Kinase Assay, Western Blot, Bradford Assay, Detection Assay, Marker, Membrane

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Transduction

Journal: Journal of Virology

Article Title: Vaccinia Virus BBK E3 Ligase Adaptor A55 Targets Importin-Dependent NF-κB Activation and Inhibits CD8 + T-Cell Memory

doi: 10.1128/JVI.00051-19

Figure Lengend Snippet: A55 inhibits NF-κB activation in a cullin-3-independent manner via its Kelch domain. Shown are immunoblots following immunoprecipitation (IP) of cleared cell lysates from HEK293T-REx cells inducibly expressing B14, A55, or EV (A), B14 or A55 (B), or B14, A55, A55-BTB, A55-Kelch, or EV (D) at 24 h postinduction with 2 μg/ml doxycycline and lysis in NP-40 (A and D) or RIPA (B) lysis buffer. Samples were subjected to SDS-PAGE and immunoblotting with the stated antibodies. (A and D) Flag-tagged immunoprecipitation and immunoblotting for endogenous cullin-3 (CUL3). (B) Reciprocal IP with protein G-Sepharose supplemented with mouse anti-Myc using cell lysates prepared 24 h posttransfection with pCDNA-Myc- CUL3 or - CUL5 . (C) Schematic of A55 domains. Full-length A55 from amino acid 1 to 565 was divided into the N-terminal BTB-BACK-containing domain and the C-terminal Kelch domain as depicted. (E) Flag IP as described for panel D in RIPA buffer using the pCW57 HEK cell lines expressing B14, A55, A55-BTB, or A55-Kelch and blotting for endogenous KPNA2. Input, cleared lysate; IP, immunoprecipitate; IB, immunoblot; *, antibody heavy/light chain. (F) HEK293T cells were transfected with pLuc-NF-κB and pRL-TK together with 100 ng of pcDNA3-Flag-KLHL12, 20 ng of pcDNA4-coB14R-Flag, of 100 ng of pcDNA4/TO-nTAP-coA55R, pcDNA4/TO-nTAP-coA55R-BTB, pcDNA4/TO-nTAP-coA55R-Kelch, or pcDNA4/TO-EV. In the experiment shown in the right panel, cells were also transfected with plasmid expressing TRAF6. After 24 h cells were either left unstimulated or stimulated with 15 ng/ml IL-1β for 6 h, and the luciferase and Renilla activities were measured. Statistical significance compared results with EV (stimulated) to those with the test samples. (G). Lysates from cells treated (as described for the left panel of F) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Data shown in all panels are representative of three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: pcDNA3-Flag- CUL3 , , , Addgene, 19893.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Expressing, Lysis, SDS Page, Transfection, Plasmid Preparation, Luciferase

Journal: Journal of Virology

Article Title: Vaccinia Virus BBK E3 Ligase Adaptor A55 Targets Importin-Dependent NF-κB Activation and Inhibits CD8 + T-Cell Memory

doi: 10.1128/JVI.00051-19

Figure Lengend Snippet: Plasmids constructed or used in this study

Article Snippet: pcDNA3-Flag- CUL3 , , , Addgene, 19893.

Techniques: Construct, Plasmid Preparation, Sequencing, Expressing, FLAG-tag, Control, Virus

Journal:

Article Title: The Human I-mfa Domain-Containing Protein, HIC, Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

doi: 10.1128/MCB.23.18.6373-6384.2003

Figure Lengend Snippet: HIC interactions in the yeast two-hybrid system

Article Snippet: Western blots were probed with anti-cyclin T1 (T-18) goat antibody or anti-CDK9 rabbit antibody (Santa Cruz Biotechnology) or with the antibodies to HA or FLAG mentioned above.

Techniques:

Journal:

Article Title: The Human I-mfa Domain-Containing Protein, HIC, Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

doi: 10.1128/MCB.23.18.6373-6384.2003

Figure Lengend Snippet: HIC interacts with P-TEFb in vivo, and this interaction is dependent upon the I-mfa domain. (A) Schematic representation of HIC and its truncations, HICΔ1 to -Δ3. The sequence of the I-mfa region (aa 165 to 246) is shown in the box, and the C termini of the Δ2 and Δ3 proteins are marked (/). (B) HIC interacts with cyclin T1 in vivo. COS cells were transfected with plasmids expressing FLAG-HIC (lane 2), FLAG-HICΔ1 (lane 3), FLAG-HICΔ2 (lane 4), FLAG-HICΔ3 (lane 5), or the FLAG tag alone (lane 1), and cell extracts prepared at 24 h posttransfection were subjected to immunoprecipitation (IP) followed by Western blotting (WB). Top, complexes immunoprecipitated with anti-FLAG antibody were resolved in a sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-cyclin T1 antibody. Bottom, as a control, 10% of the inputs used for the immunoprecipitation reactions were analyzed. (C) HIC coimmunoprecipitates with CDK9 in vivo. Transfected COS cell extracts were analyzed as for panel B except that the blot was probed with anti-CDK9 antibody. (D) HIC interacts with Tat in vivo. COS cells cotransfected with HA-Tat plasmid and the FLAG-HIC constructs were analyzed as for panel B except that the blot was probed for Tat with anti-HA antibody. (E) As for panel D, except that Tat-containing complexes were immunoprecipitated with anti-HA antibody and probed for HIC with anti-FLAG antibody.

Article Snippet: Western blots were probed with anti-cyclin T1 (T-18) goat antibody or anti-CDK9 rabbit antibody (Santa Cruz Biotechnology) or with the antibodies to HA or FLAG mentioned above.

Techniques: In Vivo, Sequencing, Transfection, Expressing, FLAG-tag, Immunoprecipitation, Western Blot, Plasmid Preparation, Construct

Journal:

Article Title: The Human I-mfa Domain-Containing Protein, HIC, Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

doi: 10.1128/MCB.23.18.6373-6384.2003

Figure Lengend Snippet: The I-mfa domain inhibits Tat transactivation. (A) Schematic representation of HIC and its truncations, HIC-N and HIC-Imfa. (B) The HIC I-mfa domain interacts with P-TEFb in vitro. Total HeLa cell extract was incubated with the indicated GST fusion proteins or with GST itself, as described by Hoque et al. (17). Bound proteins were examined by Western blotting (WB) with antibody directed against cyclin T1 (top panel) or CDK9 (bottom panel). (C) HIC I-mfa-P-TEFb complexes in cell extracts. COS cells were transfected with the indicated pcDNA3.1 expression vectors, and immunoprecipitation-Western analysis was conducted as described for Fig. ​Fig.2B.2B. Immunoprecipitates prepared with anti-Flag antibodies were probed with antibody against cyclin T1 (top panel), CDK9 (middle panel), or the Flag epitope (bottom panel). The second and fourth panels show Western blots of cell extract (without immunoprecipitation, equivalent to 10% of the input) probed with anti-cyclin T1 and anti-CDK9 antibodies, respectively. (D) HeLa, COS, 293, and NIH 3T3 cells were transfected with 100 ng of HIV-1 LTR-firefly luciferase, 20 ng of CMV-Renilla luciferase, 5 ng of pcDNA3.1-HA-Tat, and 2 μg of pFLAG-HIC-N or pFLAG-HIC-Imfa. The total amount of DNA was kept constant in each sample by the addition of pQW3.1FLAG empty vector. Transactivation was measured at 24 h and expressed as firefly/Renilla luciferase activity normalized to the value obtained with Tat alone. Data represent the averages from three experiments with standard errors.

Article Snippet: Western blots were probed with anti-cyclin T1 (T-18) goat antibody or anti-CDK9 rabbit antibody (Santa Cruz Biotechnology) or with the antibodies to HA or FLAG mentioned above.

Techniques: In Vitro, Incubation, Western Blot, Transfection, Expressing, Immunoprecipitation, FLAG-tag, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell reports

Article Title: Endothelial p130cas confers resistance to anti-angiogenesis therapy

doi: 10.1016/j.celrep.2022.110301

Figure Lengend Snippet: (A) Representative confocal images showing expression of VEGFR2 (green) in Bev-sensitive RF24-par and Bev-resistant RF24-Bev cells treated with VEGF only or VEGF + Bev. Scale bar, 50 μm; n = 3. (B) Expression of VEGFR2 pY1175 and pY1214 and total VEGFR2 in subcellular fractions of HPAECs. Only under VEGF-A (10 ng/mL) + Bev (5 μg/μL) treatment did the ~100-kD fragment of VEGFR2 appear together with the phosphorylated and total matured VEGFR2 (~220 kD). We used lamin A/C (LMNC) as a marker for the nuclear fraction (NER) and β-actin as a marker for whole-cell lysate (WCL) and cytoplasmic (Cyto) fractions. (C and D) Expression of Cyto p130cas and its 31-kDa nuclear fragment was observed in subcellular fractions from RF24-par cells but not from RF24-Bev cells (C). We used lamin B1 (LMNB1) as a marker for NER and β-actin as a marker for Cyto. When treated with VEGF + Bev, the 100-kDa nuclear fragment of VEGFR2 was observed only in subcellular fractions of RF24-par cells but not of RF24-Bev cells (D). Activated/cleaved caspase-10 was also observed in the Cyto and NER fractions of RF24-par cells treated with VEGF + Bev. (E and F) Representative confocal images showing co-localization of LC3B (green) and VEGFR2 (red) in RF24-par (E) or caspase-10-depleted RF24 casp10 — / — (F) cells in response to treatment with CTL, VEGF only, or VEGF + Bev. Scale bar, 50 μm; n = 3. (G) Top: co-immunoprecipitation of p130cas and LC3B or VEGFR2 in RF24-par cells treated with CTL, VEGF, or VEGF + Bev. Bottom: reciprocal immunoprecipitates of VEGFR2 and LC3B or p130cas. (H) Representative confocal images showing co-localization of LC3B and VEGFR2 or p130cas in RF24-par cells treated with CTL, VEGF only, or VEGF + Bev. Scale bar, 50 μm; n = 3. (I) Representative transmission electron microscopy images of mouse ovarian ECs (MOECs). In comparison with VEGF treatment, in which nuclei (NUs), rough endoplasmic reticulum (RER), and regular mitochondria (Ms) were visible, VEGF + B20 (murine AVA) treatment induced numerous autophagosomes (APs), lysosomes (Ly), and autolysosomes (Aly); abundant phagophores (Ph) were identified in Aly membranes. The substructure was observed under 5,000× and 50,000× magnification (n = 5).

Article Snippet: Myc-DDK (Flag)–tagged VEGFR2 (CAT#: RC219851; Origene, Rockville, MD) was stably overexpressed in RF24-par cells.

Techniques: Expressing, Marker, Immunoprecipitation, Transmission Assay, Electron Microscopy, Comparison

Journal: Cell reports

Article Title: Endothelial p130cas confers resistance to anti-angiogenesis therapy

doi: 10.1016/j.celrep.2022.110301

Figure Lengend Snippet: (A) Bevacizumab (Bev) induced enrichment of LC3B loci only in AVA-sensitive RF24-par cells, not in resistant RF24-Bev cells. Shown are representative confocal microscopy images for GFP-LC3B (green) expression in RF24-par or RF24-Bev cells in response to treatment with CTL, VEGF only, or VEGF + Bev. Scale bar, 50 μm; n = 3. Red arrows show the LC3B loci formed in the cells. (B) In vitro transfection with the pMAP-LC3B CRISPR-Cas9 construct used to knock out LC3B and insertion of pMAP LC3B homology-directed DNA repair (HDR) in RF24-par endothelial cells (ECs). (C) Expression or absence of LC3B in 3 CRISPR-Cas9 knockout (KO) cells. β-Actin was used as a loading CTL. (D) Representative confocal microscopy images of VEGFR2 (green) in RF24-par WT cells or LC3B2 CRISPR-Cas9 KO cells treated with VEGF + Bev. Scale bar, 50 μm; n = 3. (E–G) Autophagy inhibition by hydroxychloroquine (HCQ) reduced VEGFR2 internalization into the LC3B lysosomal compartment and NU in RF24 cells. RF24-par cells pre-treated with 40μM HCQ (24 h) were subjected to VEGF-A alone or Bev + VEGF-A treatment for another 48 h. (E) LC3B loci in cells were first determined by immunofluorescence staining with an anti-LC3B antibody. Scale bar, 50μm. (F) The autophagy flux in RF24-par cells with or without HCQ treatment was measured by acridine orange (AO) staining and analyzed with fluorescence-activated cell sorting (FACS). The percentage of acidic vesicle organelles (AVOs) was statistically compared between CTL and HCQ or VEGF-A + Bev and HCQ + VEGF-A + Bev; two-tailed Student’s t test. Data are expressed as mean ± SD (n = 3). (G) Representative images of confocal microscopy on RF24-par cells treated with HCQ or left untreated and stained with Hoechst (blue)/VE-cadherin(green)/VEGFR2 (red) for each group (CTL, VEGF, and VEGF + Bev). Each experiment was repeated at least three times, and representative images from 15 high-power fields in each condition are shown. Membrane VEGFR2 and VE-cadherin were present in RF24-par cells pretreated with HCQ + VEGF-A + Bev, where the nuclear VEGFR2 was minimal in comparison with the internalized VEGFR2 in and/or . Scale bar, 50 μm; n = 3.

Article Snippet: Myc-DDK (Flag)–tagged VEGFR2 (CAT#: RC219851; Origene, Rockville, MD) was stably overexpressed in RF24-par cells.

Techniques: Confocal Microscopy, Expressing, In Vitro, Transfection, CRISPR, Construct, Knock-Out, Inhibition, Immunofluorescence, Staining, Fluorescence, FACS, Two Tailed Test, Membrane, Comparison

Journal: Cell reports

Article Title: Endothelial p130cas confers resistance to anti-angiogenesis therapy

doi: 10.1016/j.celrep.2022.110301

Figure Lengend Snippet: (A) Nuclear TNKS1BP1 enrichment in RF24-par cells in response to Bev treatment. MOF, membranous fraction. β-Actin was used as Cyto CTL, p-cadherin as MOF CTL, and LMNB1 as NER CTL. (B) Expression of endothelial TNKS1BP1, shown by dual immunofluorescence staining for TNKS1BP1 (red) and CD31 (green), in ovarian tumor samples that were sensitive or resistant to AVA therapy (Bev). (C) Co-immunoprecipitation (coIP) of VEGFR2 and TNKS1BP1 in RF24-par cells under Bev treatment. The anti-VEGFR2 and anti-TNKS1BP1 immunoprecipitates were re-probed with an anti-p130cas antibody. (D) Representative confocal microscopy images showing TNKS1BP1 (green) and VEGFR2 (red) distributed into the NUs of RF24-par cells in response to Bev treatment (VEGF + Bev); this is distinctly different from the expression patterns in cells treated with CTL or VEGF only. Scale bar, 50 μm; n = 3. (E) Knockdown of TNKS1BP1 in RF24-par cells with shRNAs (A–D). (F and G) The graph shows mean numbers of SYTOX — live cells for each treatment group (F; data are expressed as mean ± SD, n = 3, p < 0.001 or not significant [ns], two-tailed Student’s t test. Notably, in cells treated with scramble shRNA, the percentage of SYTOX — viable cells was 36.3% under VEGF + Bev treatment; in cells transfected with shRNA A or C, the percentages of SYTOX — live cells were 72.61% and 84.98%, respectively, under VEGF + Bev treatment. Also shown are representative plots of SYTOX — populations from FACS analysis of RF24-par cells transfected with scramble shRNA, shRNA A, or shRNA C against TNKS1BP1 and treated with CTL, VEGF only, or VEGF + Bev (G). (H) Representative confocal images showing nuclear TNKS1BP1 and VEGFR2 in RF24-par scramble shRNA cells; VEGFR2 remained at the membrane in RF24-par shRNA-TNKS1BP1—A cells under VEGF + Bev treatment. Scale bar, 50 mm; n = 3.

Article Snippet: Myc-DDK (Flag)–tagged VEGFR2 (CAT#: RC219851; Origene, Rockville, MD) was stably overexpressed in RF24-par cells.

Techniques: Expressing, Immunofluorescence, Staining, Immunoprecipitation, Confocal Microscopy, Knockdown, Two Tailed Test, shRNA, Transfection, Membrane

Journal: Cell reports

Article Title: Endothelial p130cas confers resistance to anti-angiogenesis therapy

doi: 10.1016/j.celrep.2022.110301

Figure Lengend Snippet: (A) Representative images of immunohistochemical peroxidase staining for p130cas in ECs in normal human ovary or ovarian cancer tissue. Negative CTL represents a sample of ovarian cancer tissue used in the current study, processed for immunohistochemistry with a secondary antibody alone. (B) Kaplan-Meier curves showing disease-specific mortality estimates for individuals with ovarian cancer based on the degree of p130cas expression in the tumor-associated vasculature; p < 0.001, determined by unpaired two-sided Student’s t test. (C) Representative images of dual immunofluorescence staining for p130cas (red) and CD31 (green) in the same sets of human ovary or ovarian cancer samples. (D) Microvessel density (MVD) was calculated by averaging MVD from four random fields per sample. Data are expressed as mean ± SD; p < 0.05, determined by two-sided Student’s t test (n = 3). (E) p130cas expression in normal and tumor-associated ECs from normal human ovary and ovarian tumors was measured by quantitative real-time PCR. Data are expressed as mean ± SD. p < 0.05, determined by two-sided Student’s t test (n = 3). (F) Representative images of dual immunofluorescence staining of LC3B (green) and human VEGFR2 (red) (top panel) or CD31 (green) and human p130cas (red) (bottom panel) in advanced-stage human ovarian cancer samples. Individual A had an omentum tumor and responded to Bev-based therapy, individual B hada right ovary tumor as the primary site and inconclusive response to Bev, and individual C had a right ovary tumor and did not respond to Bev-based therapy. Hoechst staining (blue) was used to shown nuclei. (G) Schematic of internalization of p130cas/VEGFR2 fragments and initiation of EC death through binding with TNKS1BP1 in response to AVA treatment. Left: activated p130cas (phosphorylated) serves as a scaffold protein for activating the downstream FAK-mediated angiogenic processes (focal adhesion turnover, cell survival, and/ or migration/invasion) in ECs when stimulated by VEGF-A. Right: in response to treatment with Bev, membrane-tethered VEGFR2 is cleaved by caspase-10. Together with the ~31-kD p130cas fragment, the VEGFR2 fragment is internalized into LC3B-tagged APs. Next, a complex formed by the VEGFR2 and p130cas fragments and TNKS1BP1 translocates into the nucleus and initiates endothelial cell death, which leads to reduced angiogenesis, followed by inhibition of tumor growth.

Article Snippet: Myc-DDK (Flag)–tagged VEGFR2 (CAT#: RC219851; Origene, Rockville, MD) was stably overexpressed in RF24-par cells.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Binding Assay, Migration, Membrane, Inhibition

Journal: Journal of Advanced Research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Hypoxia-induced degradation of FTO inhibits its binding to RACK1. A–C, NIH/3T3 cells pre-treated with 5 mM 3-MA for 2 h were cultured for an additional 12 h under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions. Cells were then collected for IP using an FTO antibody. Input (A) or IP (B) samples were used for polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining. IP samples were transferred from the gel to a PVDF membrane and then incubated with the FTO antibody (C). D–F, NIH/3T3 cells were transfected with FTO-MYC and atg7 siRNA or scrambled control siRNA for 12 h and then cultured for an additional 12 under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions. Cells were then collected for IP using the MYC antibody. Input (D) or IP (E) samples were used for polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining. IP samples were transferred from the gel to a PVDF membrane and then incubated with the FTO antibody (F). G, Mass spectrometry was performed to identify proteins (around 25–35 kDa) interacting with FTO by analyzing a polyacrylamide gel stained with Coomassie Brilliant Blue from the IP group (NIH/3T3 cells overexpressing FTO-MYC treated with 3-MA and cultured for 12 h under hypoxia). H, Immunoblotting analysis of IP samples transferred from the gel to a PVDF membrane and incubated with antibodies against MYC, FTO, and RACK1. I, NIH/3T3 cells were co-transfected with RACK1-FLAG and with FTO-MYC (1–167 aa), FTO-MYC (168–334 aa), or FTO-MYC (335–502 aa) for 24 h, followed by IP to assess the interaction between FLAG and MYC. J–L, Analysis of FTO protein interaction sites using SPIDDER. Amino acids marked in pink (J) represent specific sites interacting with other proteins (K), and the amino acids marked in orange in the range of 335–502 were identified as potential binding sites for RACK1 protein (L). M and N: Using AlphaFold 2, based on the structural information of FTO (UniProt ID: Q8BGW1 ) and RACK1 (UniProt ID: P68040 ), a three-dimensional structural model of the FTO (M) and RACK1 (right side of N) proteins was generated. The amino acid sequence in the range of 335–502 is highlighted in deep red (M). Molecular docking of FTO and RACK1 was performed using ClusPro software, and the docking results were visualized with PyMOL, with brown representing the binding interfaces on FTO and light green representing the binding interfaces on RACK1 (N).

Article Snippet: Antibodies against ATG7 (8558), p62 (88588), MAP1LC3B (3868), cleaved Caspase-3 (9664), FLAG-tag (14793), MYC-tag (2276), HA-tag (3724), rabbit IgG (8726), and mouse IgG (5873) were obtained from Cell Signaling Technology.

Techniques: Binding Assay, Cell Culture, Polyacrylamide Gel Electrophoresis, Staining, Membrane, Incubation, Transfection, Control, Mass Spectrometry, Western Blot, Generated, Sequencing, Software

Journal: Journal of Advanced Research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: ATG7 mediates hypoxia-induced degradation of FTO protein through the ALP and the UPS pathways. A, NIH/3T3 cells were cultured under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions for 12 h, and mRNA levels of FTO were measured by qRT-PCR. B and C, NIH/3T3 cells were pre-treated with 50 μM cycloheximide (a protein synthesis inhibitor), 50 μM CQ (an inhibitor for autophagic lysosomes), or 10 μM MG132 (a proteasome inhibitor) for 2 h and then cultured under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions for 12 h. Protein levels of FTO were determined by Western blot (B) and quantified (C). D and E, NIH/3T3 cells transfected with atg7 siRNA or scrambled control siRNA for 12 h were cultured for an additional 12 h under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions. Immunoprecipitation (IP) was then performed to measure the K48 (D) or K63-linked ubiquitination (E) levels of FTO protein. E, G, NIH/3T3 cells pre-treated with 50 μM CQ or 10 μM MG132 for 2 h were cultured for an additional 12 h under hypoxic (1 % O 2 ) conditions. K48- (F) or K63-linked ubiquitination levels (G) of FTO protein were analyzed by IP. H, NIH/3T3 cells transfected with Mcherry-FTO-WT and GFP-LC3 for 12 h were cultured with or without 50 μM CQ for an additional 12 h under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions. Cells were then collected to observe the co-localization of Mcherry-FTO and GFP-LC3 using a Zeiss LSM 900 confocal microscope. The scale bar represents 5 μm. I, following transfection with ubiquitin siRNA in NIH/3T3 cells for 12 h, the cells were FTO-MYC, Ub-K63O-HA, and p62-WT-FLAG or p62-△UBA-FLAG for 12 h and then cultured for an additional 6 h under hypoxic (1 % O 2 ) conditions. IP was then performed to determine the interaction between MYC and HA. J, NIH/3T3 cells were co-transfected with FTO-MYC, p62-WT-FLAG or p62-△UBA-FLAG for 12 h and then cultured for an additional 6 h under hypoxic (1 % O 2 ) conditions. IP was then performed to determine the interaction between FLAG and MYC.

Article Snippet: Antibodies against ATG7 (8558), p62 (88588), MAP1LC3B (3868), cleaved Caspase-3 (9664), FLAG-tag (14793), MYC-tag (2276), HA-tag (3724), rabbit IgG (8726), and mouse IgG (5873) were obtained from Cell Signaling Technology.

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Transfection, Control, Immunoprecipitation, Ubiquitin Proteomics, Microscopy

Journal: Journal of Advanced Research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Binding between FTO and RACK1 suppresses RACK1-mediated apoptosis. A–C, NIH/3T3 cells were co-transfected with fto siRNA, RACK1-FLAG, pcDNA 3.1, FTO-MYC (1–167 aa), FTO-MYC (168–334 aa), or FTO-MYC (335–502 aa) for 12 h. Protein levels of cleaved Caspase-3 were measured by Western blot (A). Apoptotic rates were determined by flow cytometric analysis (B), and the percentage of apoptotic cells is shown in (C).

Article Snippet: Antibodies against ATG7 (8558), p62 (88588), MAP1LC3B (3868), cleaved Caspase-3 (9664), FLAG-tag (14793), MYC-tag (2276), HA-tag (3724), rabbit IgG (8726), and mouse IgG (5873) were obtained from Cell Signaling Technology.

Techniques: Binding Assay, Transfection, Western Blot

Journal: Journal of Advanced Research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Binding between FTO and RACK1 impedes the association of RACK1 with MTK1 and JNK1/2, thereby preventing MTK1 from phosphorylating JNK1/2. A and B, NIH/3T3 cells were co-transfected with fto siRNA, RACK1-FLAG, FTO-MYC, or truncated FTO (FTO-MYC) variants (1–167 aa, 168–334 aa, or 335–502 aa) for 12 h. Cells were then collected to determine the protein levels of FLAG, MYC, JNK1/2, and MTK1 by Western blot. Immunoprecipitation assays were performed to analyze the interactions between RACK1 and FTO, RACK1 and MTK1, and MTK1 and JNK1/2. C and D, NIH/3T3 cells transfected with atg7 siRNA (C) or FTO-WT-MYC (D) for 12 h were cultured for an additional 12 h under normoxic (21 % O 2 ) or hypoxic (1 % O 2 ) conditions. Cells were then collected to determine the protein levels of p-JNK1/2 (Thr183/Tyr185) and JNK1/2 by Western blot. E–G, NIH/3T3 cells were co-transfected with fto siRNA, RACK1-FLAG, FTO-MYC, truncated FTO (FTO-MYC) variants, or mtk1 siRNA for 12 h. Protein levels of p-JNK1/2 (Thr183/Tyr185) and JNK1/2 were determined by Western blot. H, NIH/3T3 cells transfected with pcDNA 3.1, FTO-MYC (335–502 aa), FTO-MYC (1–167 aa), or mtk1 siRNA for 12 h were cultured for an additional 24 h under normoxia. Protein levels of FLAG, MYC, p-JNK1/2 (Thr183/Tyr185), and MTK1 were measured by Western blot. Immunoprecipitation assays were performed to analyze the binding of JNK1/2 with FLAG and MTK1.

Article Snippet: Antibodies against ATG7 (8558), p62 (88588), MAP1LC3B (3868), cleaved Caspase-3 (9664), FLAG-tag (14793), MYC-tag (2276), HA-tag (3724), rabbit IgG (8726), and mouse IgG (5873) were obtained from Cell Signaling Technology.

Techniques: Binding Assay, Transfection, Western Blot, Immunoprecipitation, Cell Culture

Journal: Journal of Advanced Research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Verification of the anti-apoptotic role of the FTO-RACK1-MTK1-JNK1/2 cascade under hypoxia. A–D, NIH/3T3 cells were transfected with pcDNA 3.1, FTO-MYC (335–502 aa), FTO-MYC (1–167 aa), or mtk1 siRNA or treated with 10 μM SP600125 for 12 h and cultured for an additional 12 h or 24 h under hypoxia (1 % O 2 ). Cells exposed to hypoxia for 12 h were collected to measure the protein levels of RACK1, MYC, MTK1, JNK1/2, and cleaved Caspase-3 using Western blot analysis. Immunoprecipitation assays were conducted to analyze the binding of RACK1 with MYC, JNK1/2, or MTK1, as well as the interaction of MTK1 with JNK1/2 (A and B). Cells exposed to hypoxia for 24 h were collected to determine apoptotic rates using flow cytometric analysis (C), with the percentage of apoptotic cells shown in (D).

Article Snippet: Antibodies against ATG7 (8558), p62 (88588), MAP1LC3B (3868), cleaved Caspase-3 (9664), FLAG-tag (14793), MYC-tag (2276), HA-tag (3724), rabbit IgG (8726), and mouse IgG (5873) were obtained from Cell Signaling Technology.

Techniques: Transfection, Cell Culture, Western Blot, Immunoprecipitation, Binding Assay