Journal: bioRxiv

Article Title: Identification of Functional Noncoding RNA-encoded Proteins on Lipid Droplets

doi: 10.1101/2020.04.10.036160

Figure Lengend Snippet: A Workflow for the study. After isolation of LDs, low molecular weight proteins were enriched, digested, and the peptides were subjected to mass spectrometry analysis. The mass spectra obtained were searched against our database, constructed as previously reported. The flowchart for construction of database is adopted and modified from in our previous work. B a Quality verification of the isolated LDs. The quality of the isolated LDs from C2C12 was verified by silver staining and Western blot analysis. After isolation, the LD proteins were extracted and subjected to silver staining. Meanwhile, the marker proteins of LD (PLIN2, PLIN3, ACSL4, UBXD8 and CGI-58), endoplasmic reticulum (BIP), mitochondrion (VDAC, ATP5b), and cytosol (GAPDH) were analyzed by Western blot. TM, total membrane; Cyto, cytosol; PNS, post-nuclear supernatant. b Enrichment of LD proteins with a molecular mass less than 17 kDa. LD proteins were separated using a 12% Bis-Tris gel and the proteins below 17 kDa were excised for analysis by mass spectrometry. C Classification of LD-associated proteins derived from noncoding RNAs. The 15 putative LD-associated proteins encoded by noncoding RNAs were grouped according to transcript of origin. TEC, to be experimentally confirmed.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collections, Manassas, VA) were maintained in DMEM (Macgene Biotech., Beijing) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO 2 .

Techniques: Isolation, Molecular Weight, Mass Spectrometry, Construct, Modification, Silver Staining, Western Blot, Marker, Membrane, Derivative Assay

Journal: bioRxiv

Article Title: Identification of Functional Noncoding RNA-encoded Proteins on Lipid Droplets

doi: 10.1101/2020.04.10.036160

Figure Lengend Snippet: The localization of LDANP1 in C2C12 cells was studied using several methods. A The localization of transiently expressed-LDANP1. LDANP1-GFP was transiently expressed in C2C12 cells and treated with 100 µM OA for 12 h. Twenty-four hours post-transfection, cells were stained with LipidTOX Red and Hoechst (blue) to stain LDs and the nucleus, respectively. The cells were viewed using a confocal microscope. In the upper panel, Bar = 10 μm, except for Bar = 2 μm in the enlarged image. In lower panel, Bar = 5 μm, except for Bar = 1 μm in the enlarged image. B The localization of stably expressed-LDANP1. C2C12 cells stably expressing GFP-FLAG and LDANP1-GFP-FLAG were treated with 100 µM OA for 12 h. Then the cells were analyzed by immunofluorescence staining for GFP or FLAG with green-fluorescent signal to show the localization of stably expressed-LDANP1. Subsequently, the cells were stained with LipidTOX Red for LDs and Hoechst for nuclei. The cells were viewed using a confocal microscope. Bar = 5 μm, except for Bar = 1 μm in the enlarged image. C Localization of LDANP1 demonstrated by cell fractionation. C2C12 cells stably expressing LDANP1-GFP-FLAG and GFP-FLAG control cells were cultured in 100 µM OA supplemented medium for 12 h. Cells were harvested for cell fractionation. The proteins in different cellular fractions were separated by SDS-PAGE and analyzed by Western blot with the indicated antibodies. GF, GFP-FLAG cells; LGF, LDANP1-GFP-FLAG cells. TM, total membrane; Cyto, cytosol; PNS, post-nuclear supernatant. D Immuno-gold labeling for LDANP1. C2C12 cells stably expressing LDANP1-GFP-FLAG were treated with 100 µM OA plus 1 µM MG132 for 12 h. Then the cells were prepared for immuno-gold labeling of GFP to display the localization of LDANP1. Briefly, cells were fixed with 4% (v/v) paraformaldehyde, dehydrated in an ascending concentration series of ethanol and embedded in LR White resin. After ultra-thin sectioning, sections were stained for GFP with secondary antibody conjugated to 18 nm colloidal gold. The sections were observed by transmission electron microscope. Bar = 200 nm.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collections, Manassas, VA) were maintained in DMEM (Macgene Biotech., Beijing) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO 2 .

Techniques: Transfection, Staining, Microscopy, Stable Transfection, Expressing, Immunofluorescence, Cell Fractionation, Control, Cell Culture, SDS Page, Western Blot, Membrane, Labeling, Concentration Assay, Thin Sectioning, Transmission Assay

Journal: bioRxiv

Article Title: Identification of Functional Noncoding RNA-encoded Proteins on Lipid Droplets

doi: 10.1101/2020.04.10.036160

Figure Lengend Snippet: A Alignment of putative LDANP1 peptide sequences from a variety of mammals. LDANP1 amino acid sequences were translated from short ORF in USPL1 sequence in 11 different mammalian species. Alignment of those sequences were analyzed using BioEdit. Amino acid identity is indicated. B Schematic depicting CRISPR/Cas9-mediated LDANP1 knock-in strategy. Cells carrying a 3×FLAG-HA tag in the C-terminus of LDANP1 allele were generated by CRISPR/Cas9-mediated knock-in technique. C Verification of the insertion of the 3×FLAG-HA tag by PCR. Genomic DNA from selected clones was probed with primer pairs designed to amplify 200 bp upstream and 200 bp downstream of the LDANP1 stop codon. The PCR amplicons were subjected to 2% (w/v) agarose gel electrophoresis. D Verification of endogenous translation of LDANP1. LDANP1 knock-in cell line and WT C2C12 cells were treated with or without 1 µM MG132 for 12 h. Cells were washed with PBS, lysed with 1 mL TETN solution and probed with anti-FLAG beads to precipitate FLAG-tagged protein. The precipitates were eluted in sample buffer, separated on 5-15% gradient SDS-PAGE, and analyzed by FLAG Western blot (upper panel). A silver-stained gel (lower panel) indicated the protein profile in each sample and served as protein loading control. IP, immunoprecipitation.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collections, Manassas, VA) were maintained in DMEM (Macgene Biotech., Beijing) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO 2 .

Techniques: Sequencing, CRISPR, Knock-In, Generated, Clone Assay, Agarose Gel Electrophoresis, SDS Page, Western Blot, Staining, Control, Immunoprecipitation

Journal: bioRxiv

Article Title: Identification of Functional Noncoding RNA-encoded Proteins on Lipid Droplets

doi: 10.1101/2020.04.10.036160

Figure Lengend Snippet: A Reduction of triacylglycerol level by LDANP1. Stable cells expressing LDANP1-GFP-FLAG and GFP-FLAG control cells were cultured to confluence in 6-well dishes and treated with or without 100 µM oleate (OA) for 12 h. Cells were collected in PBS (pH 7.4) containing 1% (v/v) Triton X-100 and sonicated. An aliquot of cell lysate was analyzed for triacylglycerol (TAG) using total protein as internal control. Data represent mean ± SEM (n = 4), and were analyzed by unpaired Student t -test. **, P<0.001. GF, GFP-FLAG cells; LGF, LDANP1-GFP-FLAG cells. B Inhibitory effect of LDANP1 on insulin sensitivity in C2C12 cells. Cells were incubated in the presence or absence of 100 μM OA with or without 500 μM palmitate (PA) for 12 h before insulin stimulation (200 nM insulin for 10 min at 37°C). Cells were then lysed with sample buffer and the proteins were subjected to Western blot analysis of p-Akt and GAPDH. LGF, LDANP1-GFP-FLAG cells.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collections, Manassas, VA) were maintained in DMEM (Macgene Biotech., Beijing) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO 2 .

Techniques: Expressing, Control, Cell Culture, Sonication, Incubation, Western Blot

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Schematic description of substrate identification with PRMT1 Y39FM48G and Pob-SAM using BPPM technology. ( B ) HEK293T cells were transfected with either empty vector or the PRMT1 Y39FM48G mutant, followed with treatment of Adox to induce hypomethylation. These cells were then lysed to release the PRMT1 substrates as previously described and the cell lysates were treated with Pob-SAM cofactor. The terminal alkyne-modified substrates were conjugated with the cleavable azido-azo-biotin probe, followed by streptavidin enrichment, sodium dithionite elution and western blotting detection. ( C ) Western blotting analysis of the pull-down substrate of PRMT1. The eluted targets of PRMT1 were incubated with RBM15 antibody. The pull-down sample from 293T cells transfected with PRMT1 Y39FM48G variant showed significantly higher level of RBM15 compared to the control group. Cell lysates without streptavidin enrichment were assessed by anti-RBM15 western blotting as loading control (Input panel). BPPM, bio-orthogonal profiling of protein methylation; Pob-SAM, propargyloxy-but-2-enyl-S-adenosylmethionine; PRMTs, protein arginine methyltransferases DOI: http://dx.doi.org/10.7554/eLife.07938.005

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Modification, Western Blot, Incubation, Variant Assay, Control, Methylation

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Tandem mass spectrometry analysis for Flag-tagged RBM15 purified from 293T cells overexpressing RBM15-Flag. The arrow indicates the modified peptide. ( B ) Schematic diagram shows domains on RBM15. ( C ) Dot blot to test the antibody (Mono-R100 from Cell Signaling, Danvers, MA) recognizing monomethylated RBM15 peptide based on mass spectrometry analysis. Nitrocellulose membrane was spotted with peptides with no modification, with arginine mono-methylated or with arginine asymmetrically dimethylated. The peptide sequences were listed on the right side of the dot blot gel. ( D ) Dot blot to test the antibody (D4H5 from Cell Signaling) which recognizes asymmetrically dimethylated RBM15 peptide. Peptides were spotted in the same order as in panel C. ( E ) IP-WBwestern blot for RBM15 protein in Meg-01 cell lines expressing Flag-tagged RBM15 and Flag-tagged RBM15 R578K. Anti-monomethyl arginine antibody was used for immunoprecipitation and the ectopically expressed RBM15 proteins were detected by Flag antibody. ( F ) IP-western blotting assays for detecting methylated RBM15 and R3K mutant expressed in 293T cells. RBM15-R3K stands for RBM15 protein with all the arginines in the LYRDRDRD sequence mutated to lysines. ( G ) The in vitro methylation reaction with peptide (LYRDRDRDLY) incubated with purified PRMT1. H4 peptide (20 mer) was used as a positive control. The methylated peptide was detected by the D4H5 dimethyl arginine antibody not by the mono-methyl antibody. IP, immunoprecipitation protocol; PRMTs, protein arginine methyltransferases; SPOC, spen paralog and ortholog C-terminal. DOI: http://dx.doi.org/10.7554/eLife.07938.006

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Mass Spectrometry, Purification, Modification, Dot Blot, Membrane, Methylation, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Sequencing, In Vitro, Incubation, Positive Control

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Alignment of RBM15 sequences among different species shows R578 within a conserved protein region. ( B ) RBM15-Flag and its mutant (R578K), affinity purified with anti-Flag antibody from transfected 293T cells, were detected by WB with anti-monomethyl arginine antibody. ( C ) Flag-tagged RBM15 was affinity purified by Flag antibody for WB with two generic antibodies against mono-methyl arginine and dimethyl arginine. The 293T cells overexpressing wild type RBM15-Flag protein with PRMT1 V2 (lane 2) and V1 (lane 3) were treated with 20nM MG132 for 6 hr before harvesting. ( D ) The differences between N terminal sequences of PRMT1 V1 and V2 isoforms. ( E ) In vitro methylation assays. Affinity purified RBM15 protein was methylated by incubation with purified HA-PRMT1 and 0.15 mM of S-adenosyl-methionine at 30°C for 4 hours. The methylated RBM15 was detected by anti-dimethyl arginine antibody in WB. ( F ) RBM15 was immunoprecipitated with a mouse monoclonal anti-RBM15 antibody from whole cell extract prepared from a MEG-01 stable cell line expressing inducible short hairpin RNA against PRMT1. Normal IgG was used as immunoprecipitation controls. The immunoprecipitated RBM15 was detected by WB with antibodies against mono-methyl arginine (mono-R100) and asymmetrical dimethyl arginine (D4H5). As controls, we did WB with anti-PRMT5 and anti-PRMT4 antibodies. ( G ) Detection of methylated RBM15 in MEG-01-stable cell lines expressing Flag-RBM15 and R578K mutant proteins after a PRMT1 inhibitor (DB75) treatment for 24 hr. RBM15 is affinity purified by anti-Flag antibody and detected by WB with mono-methyl arginine antibody and dimethyl arginine antibody. PRMT, protein arginine methyltransferases; WB, western blot. DOI: http://dx.doi.org/10.7554/eLife.07938.003

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Mutagenesis, Affinity Purification, Transfection, In Vitro, Methylation, Incubation, Purification, Immunoprecipitation, Stable Transfection, Expressing, shRNA, Western Blot

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Dot blot for the anti-methyl-RBM15 antibody. Synthesized RBM15 peptides with different methylation status on arginine 578 were spotted for blotting with the anti-methyl RBM15 antibody. ( B to D ) RBM15 methylation status was measured by anti-methyl-RBM15 antibody by western blotting. ( B ) The whole cell extracts from 293T cells expressing PRMT1 were used for western blotting with the anti-methyl RBM15 antibody. Empty vector was used as a control. ( C ) The whole cell lysates from 293T cells expressing WT RBM15 or R578K mutant were used for western blotting. ( D ) A stable MEG-01 cell line expressing Dox-inducible shPRMT1 was used to knock down PRMT1. The expression of methyl-RBM15 was measured by western blotting with anti-methyl RBM15 antibody, RBM15 antibody, PRMT1 antibody and GAPDH antibody. Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRMTs, protein arginine methyltransferases; WT, wild type DOI: http://dx.doi.org/10.7554/eLife.07938.007

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Dot Blot, Synthesized, Methylation, Western Blot, Expressing, Plasmid Preparation, Control, Mutagenesis, Knockdown

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) WB for RBM15 in 293T cells overexpressing PRMT1 V1 and V2. PRMT1 V2 was detected by anti-V2 specific antibody (PRMT1 V2). PRMT1 V1 and V2 were detected by an antibody for all isoforms (labeled as PRMT1). ( B ) The level of the RBM15 protein as detected by WB in MEG-01 cells treated with methyltransferase inhibitors (Adox and MTA mix) or DB75. ( C ) RBM15 protein level was measured by WB in MEG-01 cells with two doxycycline-inducible shRNA against PRMT1 (on the left). In the middle and right sides are real-time PCR results to show the mRNA levels of total amount of PRMT1, PRMT1 V2, and RBM15 in shPRMT1#1 stable MEG-01 cell line (middle) and in shPRMT1#2 stable MEG-01 cells (right). All data are presented as mean ± standard deviation from three independent experiments. ( D ) RBM15 protein level was measured by WB in MEG-01 cells induced by Dox to express PRMT1 V2 isoform. On the right are the real-time PCR charts for PRMT1 V2 and RBM15 mRNA levels. Data are presented as mean ± standard deviation from three independent experiments. ( E ) RBM15 protein level was accessed by WB in a MEG-01 stable cell line expressing shRNA against V2. The names of antibodies are listed on right. The pRS vector retrovirus infected MEG-01 cells were used as control. ( F ) WB with anti-Flag antibody to detect the protein levels of RBM15 wild type and R578K mutant proteins in 293T cells overexpressing PRMT1 V2 and RBM15 proteins. ( G ) The half-life of the RBM15 proteins in MEG-01 cells, and stable cell lines overexpressing Flag-tagged RBM15 and RBM15 R578K were assessed by WB. Cyclohemixide were added to stop protein synthesis 30 min before harvesting cells as the 0 time point. The half-life curves were plotted by GraphPad Prism 6. Adox, adenosine dialdehyde; DMSO, dimethyl sulfoxide; Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; MTA, methylthioadenosine; PCR; polymerase chain reaction; PRMTs, protein arginine methyltransferases; shRNA; short hairpin RNA; WB, western blot. DOI: http://dx.doi.org/10.7554/eLife.07938.008

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Labeling, shRNA, Real-time Polymerase Chain Reaction, Standard Deviation, Stable Transfection, Expressing, Plasmid Preparation, Infection, Control, Mutagenesis, Polymerase Chain Reaction, Western Blot

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) WB for the RBM15 protein from MEG-01 cells treated with the proteasome inhibitor MG132. ( B ) The ubiquitylated RBM15-Flag was detected by anti-Flag antibody. The poly-ubiquitylated RBM15 was purified with nickel beads under denaturing conditions (6M of guanidine-HCl) from 293T cells expressing RBM15-Flag and poly-histidine-tagged ubiquitin. ( C ) The ubiquitylated RBM15 was measured by anti-ubiquitin antibody after affinity purified with Flag antibody from MG132 treated 293T cells expressing RBM15-Flag or R578K-Flag and ubiquitin. ( D ) IP-WB for poly-ubiquitylated RBM15 in DB75 treated MEG-01 cells. The endogenous RBM15 protein was immunoprecipitated by anti-RBM15 antibody and then blotted with anti-ubiquitin antibody and anti-RBM15 antibody. ( E ) The ubiquitylated RBM15 was detected by anti-Ub antibody after RBM15-Flag as well as its mutant was affinity purified from 293T cells transfected with combinations of plasmids shown above the gel. CNOT4 was detected via its HA tag. ( F ) WB to detect RBM15 protein levels in two MEG-01 cell lines expressing two different shCNOT4. PRMT1 inhibitor (DB75) was used to treat the cells expressing shCNOT4 RNAs. The efficiency of shCNOT4 knockdown was checked by real-time PCR. ( G ) In vitro ubiquitylation assays with CNOT4 and RBM15. Purified PRMT1 was added to methylate RBM15 in vitro in lanes 5 and 6 first before incubating with CNOT4 for in vitro ubiquitylation assays. All components were affinity purified from 293T cells. The ubiquitylated RBM15-Flag was detected by WB with anti-Flag antibody. ( H ) CNOT4 from MEG-01 whole cell extract was pulled down with methylated and nonmethylated peptides of RBM15. CNOT4 was detected by WB with anti-CNOT4 antibody. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; IP, immunoprecipitation protocol; PCR, polymerase chain reaction; PRMTs, protein arginine methyltransferases; WB, western blot. DOI: http://dx.doi.org/10.7554/eLife.07938.009

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Purification, Expressing, Ubiquitin Proteomics, Affinity Purification, Immunoprecipitation, Mutagenesis, Transfection, Knockdown, Real-time Polymerase Chain Reaction, In Vitro, Methylation, Polymerase Chain Reaction, Western Blot

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Real-time PCR assays for CNOT4 in MEG-01 cells expressing shCNOT4. ( B ) WBs show the knockdown efficiencies of two shRBM15 constructs in MEG-01 cells. ( C ) Using CRISPR to knockdown CNOT4 in 293T cells (A5 is a 293T cell line with only one wild type allele of CNOT4) as shown by real-time PCR on the left and by WB on the right. RBM15 protein level goes up in CNOT4 +/-− cells by WB assays. CRISPR, clustered regularly interspaced short palindromic repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; PCR, polyerase chain reaction; WB, western blot. DOI: http://dx.doi.org/10.7554/eLife.07938.011

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Knockdown, Construct, CRISPR, Western Blot

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) The Flag-tagged RBM15 protein was affinity purified by anti-Flag antibody from 293T cells overexpressing FLAG RBM15 protein as shown by Coomassie staining of 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. ( B ) Affinity purified HA-CNOT4 and HA-PRMT1 proteins were shown by silver staining. Both proteins were produced in 293T cells transfected with pCDNA3-HA-PRMT1 and pCDNA3-HA-CNOT4, respectively. Anti-HA antibody (12Ca5) column was used to purify these proteins. Proteins were eluted by 0.5mg/ml of HA peptide (Sigma) in PBS. HA, hemagglutinin; PBS, phosphate-buffered saline; PRMTs, protein arginine methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.07938.010

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Affinity Purification, Staining, Polyacrylamide Gel Electrophoresis, Silver Staining, Produced, Transfection, Saline

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A-C ) The relative expression levels of total PRMT1 (including V1 and V2) ( A ), PRMT1 V2 ( B ) and RBM15 ( C ) in different mouse lineages were measured by real-time polychromase chain reaction. The expression level is normalized to GAPDH, then normalized to the level in LT-HSC. ( D ) The surface markers we used to sort mouse lineages. ( E ) The expression level of PRMT1 in different human hematopoietic lineages. ( F ) The expression level of RBM15 in different human hematopoietic lineages based on database from HemaExplorer. ( G ) The abbreviations for the different human hematopoietic lineages used in panels ( E ) and ( F ). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LT-HSC, long-term hematopoietic stem cells; PRMTs, protein arginine methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.07938.013

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Expressing

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) WB to measure the protein levels in MEG-01 cells stimulated to maturation by PMA. The left panel (WB results) showed the protein levels by antibodies against GAPDH, RBM15, methyl-RBM15, PRMT1 with a PRMT1 antibody against all isoforms and PRMT1 V2 with specific V2 antibody during the course of maturation. The middle panel shows the quantitation of the protein bands in the WBs on the left normalized to GAPDH. The right panel showed the decrease of PRMT1 V2 by real-time PCR during maturation with GAPDH mRNA as an internal control. Real-time PCR data were presented as means ± standard deviation from three independent experiments. ( B ) Histograms of CD41 + cells on PMA-treated MEG-01 cells overexpressing RBM15 and RBM15R578K mutant proteins on day 3. The percentage of CD41 + cells was calculated according to matched antibody isotype control. Three independent experiments were done with statistics shown on the left. P*** <0.001, P** <0.01. ( C ) FACS analysis of the polyploid status of PMA-treated cells overexpressing RBM15 and R578K mutant proteins by PI staining. Vector: lentivirus vector. P*** <0.001. ( D ) The matured MK cells were measured by CD61 + CD42 + . Human adult CD34 + cells in pro-MK differentiation medium were treated with DB75 for 3 days. Three independent experiments were done with P*< 0.05. ( E ) Human adult CD34 + cells were infected with lentivirus expressing PRMT1 V2 or lentivirus vector and grown in pro-MK differentiation medium for 5 days. Three independent experiments were done with P***<0.001. ( F ) Human adult CD34 + cells were infected with two lentiviruses expressing shRNAs against RBM15 and grown in pro-MK differentiation medium. Three biological replicates were used for P value. P** <0.01. ( G ) Human adult CD34 + cells were infected with lentiviruses expressing RBM15 or R578K proteins and grown in pro-MK differentiation medium for 5 days. Three independent experiments were done with P**<0.01. ( H ) Human adult CD34 + cells were infected with lentiviruses expressing RBM15 or R578K together with a lentivirus expressing PRMT1 V2 and grown in pro-MK differentiation medium. Three biological replicates were used for P value. P**<0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI, propidium iodide; PMA, phorbol myristate acetate; PRMTs, protein arginine methyltransferases; WB, western blot; WT, wild type. DOI: http://dx.doi.org/10.7554/eLife.07938.012

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction, Control, Standard Deviation, Mutagenesis, Staining, Plasmid Preparation, Infection, Expressing, Western Blot

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Real-time PCR assays for detecting RNA associated with RBM15 in MEG-01 cells by RIP with the RBM15 antibody. The levels of RBM15-associated mRNAs were calculated as mean ± standard deviation from three independent experiments. ( B ) The distribution of RBM15 binding sites. All the RBM15 target genes were listed in . ( C ) GO pathway analysis (FDR<0.01) showed pathways associated with genes that have RBM15 binding sites in introns. ( D ) GO pathway analysis (FDR <0.01) revealed pathways associated with genes containing RBM15 binding sites in 3’UTR regions. ( E ) Differential exon usage events detected by the MISO program. ( F ) The changes of percentage splice-in events in different splicing categories when RBM15 is knocked down. ( G ) MISO plot for skipping of GATA1 exon 2 when RBM15 was knocked down. ( H ) Isoforms of GATA1fl and GATA1s were detected by PCR using RNA extracted from MEG-01 cells with or without RBM15 knockdown. ALE, alternative last exon; AFE, alternative first exon; A5SS, alternative 5’ splicing sites; A3SS, alternative 3’ splicing sites; GO, gene ontology; MXE, mutually exclusive exon usage; PCR, polymerase chain reaction; RI, retention intron; RIP, RNA immunoprecipitation assay; SE, skipped exon; T3UTR, tandem UTR. DOI: http://dx.doi.org/10.7554/eLife.07938.015 10.7554/eLife.07938.016 Figure 5—source data 1. Identification of RNAs associated with RBM15 by RNA immunoprecipitation assay with anti-RBM15 antibody. Genes related to MK differentiation are highlighted. DOI: http://dx.doi.org/10.7554/eLife.07938.016 10.7554/eLife.07938.017 Figure 5—source data 2. Analysis of gene expression profile changes with RNA-seq data from RBM15 knockdown MEG-01 cells. Genes related to MK differentiation are highlighted. MK, megakaryocyte; RNA-seq, RNA sequencing. DOI: http://dx.doi.org/10.7554/eLife.07938.017 10.7554/eLife.07938.018 Figure 5—source data 3. Analysis of differential exon usage regulated by RBM15 with RNA-seq data from RBM15 knockdown MEG-01 cells. Genes related to MK differentiation are highlighted. MK, megakaryocyte; RNA-seq, RNA sequencing. DOI: http://dx.doi.org/10.7554/eLife.07938.018

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay, Knockdown, Polymerase Chain Reaction, RNA Immunoprecipitation, Gene Expression, RNA Sequencing

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) RBM15 binding peaks on pre-mRNA of CDC42. ( B ) RBM15 binding sites on pre-mRNA of macroH2A (H2AFZ). ( C ) RBM15 binding on pre-mRNA of TAL1. ( D ) RBM15 binding peaks on pre-mRNA of LEF1. mRNA, messenger RNA. DOI: http://dx.doi.org/10.7554/eLife.07938.019

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Binding Assay

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: In the MISO diagram, the red graph is for MEG-01 cells, and the orange diagrams are for two MEG-01 cell lines expressing two shRBM15 RNAs. ( A ) TAL1 (aka SCL) skips exon 2 and 3 as detected by DEXSeq and use alternative short 3’UTR after RBM15 was knocked down. TAL1 has the exon 2 (1:47691115–47691561) skipped in RBM15 knockdown cells, which losses protein coding ability for transcript (bottom panel). ( B ) MacroH2A (H2AFZ) retains intron (4:100870543–100870820) when RBM15 protein level is reduced. ( C ) RUNX1 has multiple exon usage alteration events as demonstrated by DEXSeq on the top. The E017 marks the change for generating RUNX1a (E7a is included) when RBM15 is knocked down. The bottom is MISO analysis showing that Exon 6 is skipped to generate a RUNX1 protein without the transcriptional repression region. This isoform was reported . ( D ) CDC42 skips the exon 2a (1:22400587–22400712) or exon 2b (1:22400647–22400712). Although the open reading frame is still intact with the skipping events, the change of 5’UTR might cause changing in efficiency of protein translation. DOI: http://dx.doi.org/10.7554/eLife.07938.022

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Expressing, Knockdown

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) Alternative splicing of RUNX1, GATA1, c-MPL in MEG-01 cells and MEG-01-derived stable cell lines overexpressing RBM15 and PRMT1 V2 or knocking down RBM15 and PRMT1 V2. The ratios of different isoforms were calculated from real-time PCR assays with isoform specific primers. At least three independent experiments were performed. P****: <0.01; P***: <0.05; P**: <0.2 and P*: <0.3 compared to their respective vector control groups. ( B ) Time course for alternative splicing of RUNX1, GATA1 and c-MPL in human adult CD34 + cells grown in pro-MK differentiation medium. Three independent experiments were used to calculate the standard deviation. ( C ) The alternative splicing of GATA1, RUNX1 and c-MPL was measured as ratio change in human adult CD34 + cells treated with DB75 overnight in basic cytokine mix. Three independent experiments were used to calculate the P values. PCR, polymerase chain reaction; PRMT, protein arginine methyltransferase DOI: http://dx.doi.org/10.7554/eLife.07938.023

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Alternative Splicing, Derivative Assay, Stable Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Standard Deviation, Polymerase Chain Reaction

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: The data are the averages from three independent experiments with standard deviation. ( A ) the ratio between c-MPL72minus-exon9/c-Mpl-exon9+. ( B ) the ratio between c-MPL-tr/c-MPL-exon 9+. The c-MPL-tr stands for c-MPL isoform without exon 9 and exon 10. We also observed that the ratio of the c-MPL isoform with 72bp missing in the exon 9 (i.e. c-MPL-72minus-exon9+) to c-MPL-exon 9+ mRNA is similarly changed like the ratio of c-MPL-exon 9-/c-MPL-exon 9+ mRNA upon overexpression or knockdown of RBM15 or of PRMT1. We discovered that the ratio of c-MPL-tr (which misses both exon 9 and exon 10) to c-MPL-exon9+ was changed likewise when the RBM15 level is changed as shown in . Dox, doxycycline; PCR, polymerase chain reaction DOI: http://dx.doi.org/10.7554/eLife.07938.026

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Standard Deviation, Over Expression, Knockdown, Polymerase Chain Reaction

Journal: eLife

Article Title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

doi: 10.7554/eLife.07938

Figure Lengend Snippet: ( A ) The interaction between SF3B1 and RBM15 in the context of PRMT1-mediated methylation. RBM15-Flag and RBM15 R578K-Flag expressed from two MEG-01 cell lines with or without DB75 treatment were immunoprecipitated with anti-Flag antibody for detecting interaction with SF3B1 by WB. ( B ) The endogenous SF3B1 was co-immunopreciptiated with anti-RBM15 antibody in MEG-01 cells expressing inducible shPRMT1. Normal mouse serum was used as a negative control. ( C ) RBM15 binding profile on GATA1 pre-mRNA based on RIP-seq data. The green peaks are the binding sites for RBM15 and the blue profile is the binding profile for normal IgG. Two biological replicates were used for bioinformatic analysis. The significant peaks were shaded with pink squares. ( D ) The regions where RBM15 (RIP with RBM15 antibody, left panel) and SF3B1 (RIP with SF3B1 antibody right panel) bound on GATA1 pre-mRNA in MEG-01 cells (solid bar) and RBM15 knockdown MEG-01 cells (open bar) were mapped by real-time PCR assays. The locations of primers on the pre-mRNA of GATA1 were shown on the bottom. Three biological replicates were used to calculate the standard deviations. GAPDH intron 1 was used as negative controls for both antibodies. ( E ) The regions on c-MPL pre-mRNA, where RBM15 and SF3B1 bound in MEG-01 cell lines expressing shRBM15 (square line) or expressing pLKO vector (solid dot line), were assessed by RIP with RBM15 (left panel) and SF3B1 antibodies (right panel). The locations of primers on the pre-mRNA of c-MPL are shown on the bottom. Three biological replicates were used for standard deviations. ( F ) A model for RBM15-mediated regulation of alternative RNA splicing. RBM15 and SF3B1 cooperate to produce GATA1fl and low level of RBM15 leads to lower SF3B1 binding and skipping of the exon 2. PRMT1-mediated methylation of RBM15 controls the ubiquitylation of RBM15 by CNOT4, thus controlling the balance between proliferation and differentiation in megakaryopoiesis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; RIP, RNA immunoprecipitation assay; PCR, polymerase chain reaction; PRMT, protein arginine methyltransferase DOI: http://dx.doi.org/10.7554/eLife.07938.027 10.7554/eLife.07938.028 Figure 7—source data 1. Mass spectrometry analysis of RBM15-associated proteins. DOI: http://dx.doi.org/10.7554/eLife.07938.028

Article Snippet: The other antibodies used were commercially available, including anti-Flag M2 Ab (#F1804, Sigma), RBM15 monoclonal antibody (#66059-1-1g, Proteintech, Chicago), RBM15 polyclonal antibody (#: 10587-1-AP, Proteintech, Chicago), PRMT1 (#07404, Upstate Biotechnology), SF3B1 (#PA5-19679, Thermo Scientific), Ub (#U5397, Sigma) and GAPDH (#MA5-15738, Thermo Scientific, Waltham).

Techniques: Methylation, Immunoprecipitation, Expressing, Negative Control, Binding Assay, Knockdown, Real-time Polymerase Chain Reaction, Plasmid Preparation, RNA Immunoprecipitation, Polymerase Chain Reaction, Mass Spectrometry

Journal: Scientific Reports

Article Title: Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

doi: 10.1038/s41598-018-30253-8

Figure Lengend Snippet: Cdc14A is a phosphoprotein with autodephosphorylation ability. ( a) HEK293T were transiently transfected to express Flag-Cdc14A or its inactive form Flag-Cdc14A(PD). After 24 hours, cells were treated with OA (0,5 μM), or not, during 2 hours. Cellular lysates were analyzed by immunoblot to detect the indicated proteins. Activation of Cdk1, as a consequence of OA treatment, was confirmed by phospho-Cdk1(Tyr15) detection. Phos-tag gel was used to specifically identify the phosphorylation status of the Cdc14A forms. (b) Total protein extracts from HEK293T cells transfected with Flag-Cdc14A or Flag-Cdc14A(PD), as described in (a), were incubated with or without lambda phosphatase (λPP), resolved by Phos-tag gels and analyzed by immunoblot with anti-Cdc14A antibodies. Full-length blots are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used were mouse HA-tag (12CA5, Ref. 11-666606001, Boehringer Mannheim), mouse Flag-tag (M2, peroxidase conjugated, Ref. A8592, Sigma) or Rat anti-Flag, (Ref. 299474, Agilent Technologies), mouse Myc-tag (9E10, Ref. M5546, Sigma), mouse Cdk1 (Ref. sc-54, Santa Cruz Biotechnology), rabbit phospho-Tyr15-Cdk1 (Ref. 9111, Cell Signalling), rabbit phospho-(Ser) CDKs (Ref. 2324, Cell Signaling), goat Cdc14A (N-18, Ref. sc-25952, Santa Cruz Biotechnology), rabbit Cyclin B1 (Ref. sc-752, Santa Cruz Biotechnology), mouse PP2A (ID6, Ref. 05-421, Millipore), rabbit Wee1 (Ref. sc-9037, Santa Cruz Biotechnology), mouse Plk1 (Ref. 33-1700, Zymed), rabbit phospho-histone-H3(S10) (Ref. 06-570, Millipore) and mouse β-Actin (AC-15, Ref. A5441, Sigma).

Techniques: Transfection, Western Blot, Activation Assay, Phospho-proteomics, Incubation

Journal: Scientific Reports

Article Title: Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

doi: 10.1038/s41598-018-30253-8

Figure Lengend Snippet: Cdk1-dependent phosphorylation of Cdc14A early in mitosis. ( a) U-2-OS cells stably expressing HA-Cdc14A were synchronized at G1/S transition by a double thymidine block (0 hr) and then released into fresh medium containing nocodazole (NZ) to allow progression through G2 phase and G2/M transition and to avoid mitotic exit. For mitosis-synchronized cells, the cultures were treated with nocodazole during 12 hours and rounded mitotic cells were collected (0 hr) or released into fresh medium to allow progression through mitosis and G1 phase. Phosphorylation state of Cdc14A was analyzed by immunoblot, using Phos-tag gels, at the indicated time points. Progression through the cell cycle was monitored by immunoblot against the indicated proteins. (b) U-2-OS-HA-Cdc14A cells were treated with nocodazole during 12 hours. Rounded mitotic cells were selected by shake-off and then treated with the vehicle DMSO (dimethyl sulfoxide) or the Cdk1 inhibitors Purvalanol A (Pur) and RO-3306 (RO) during 3,5 hours. Samples were analyzed by immunoblot with the indicated antibodies. Asynchronous cells were also analyzed, and phosphorylation state of HA-Cdc14A was checked using Phos-tag gels. (c) GST-Cdc14A, (wt), and GST-Cdc14A(PD), (PD), were phosphorylated in vitro by Cdk1-Cyclin B1 complexes in the presence (+) or absence (−) of 1 mM sodium orthovanadate (V), using γ( 32 P)ATP. Reactions were resolved in SDS-PAGE gels; proteins were visualized by Coomassie staining and phosphorylation level was analyzed by autoradiography. Left panel corresponds to a representative experiment. The 32 P incorporation level was normalized by the amount of recombinant protein. The 32 P signal of GST-Cdc14A wt and GST-Cdc14A(PD) in the presence of V was considered 100%. Bars show the quantification from three independent experiments. Full-length of blots and gels are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used were mouse HA-tag (12CA5, Ref. 11-666606001, Boehringer Mannheim), mouse Flag-tag (M2, peroxidase conjugated, Ref. A8592, Sigma) or Rat anti-Flag, (Ref. 299474, Agilent Technologies), mouse Myc-tag (9E10, Ref. M5546, Sigma), mouse Cdk1 (Ref. sc-54, Santa Cruz Biotechnology), rabbit phospho-Tyr15-Cdk1 (Ref. 9111, Cell Signalling), rabbit phospho-(Ser) CDKs (Ref. 2324, Cell Signaling), goat Cdc14A (N-18, Ref. sc-25952, Santa Cruz Biotechnology), rabbit Cyclin B1 (Ref. sc-752, Santa Cruz Biotechnology), mouse PP2A (ID6, Ref. 05-421, Millipore), rabbit Wee1 (Ref. sc-9037, Santa Cruz Biotechnology), mouse Plk1 (Ref. 33-1700, Zymed), rabbit phospho-histone-H3(S10) (Ref. 06-570, Millipore) and mouse β-Actin (AC-15, Ref. A5441, Sigma).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Blocking Assay, Western Blot, In Vitro, SDS Page, Staining, Autoradiography, Recombinant

Journal: Scientific Reports

Article Title: Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

doi: 10.1038/s41598-018-30253-8

Figure Lengend Snippet: Cdc14A both autodephosphorylates and is dephosphorylated by other phosphatase(s) at the exit from mitosis. ( a ) U-2-OS cells stably expressing the inactive form HA-Cdc14A(PD) were synchronized as described in Fig. . Phosphorylation state of the inactive form of Cdc14A was analyzed by immunoblot, using Phos-tag gels, at the indicated time points. Progression through the cell cycle was monitored by immunoblot against the indicated proteins. (b) GST-Cdc14A(PD) was phosphorylated in vitro by Cdk1-Cyclin B1 complexes in the presence of γ( 32 P)ATP. Samples were then washed and incubated with buffer alone or with 100 ng of GST-Cdc14A, GST-Cdc14A(PD), GST-Cdc14B or GST-Cdc14B(PD). Left panel corresponds to a representative experiment. The 32 P incorporation levels were normalized by the amount of recombinant. The 32 P signal of GST-Cdc14A(PD) in the presence of buffer alone was considered 100%. Bars show the quantification from three independent experiments. (c) GST-Cdc14B, (wt), and GST-Cdc14B(PD), (PD), were phosphorylated in vitro by Cdk1-Cyclin B1 complexes in the presence of γ( 32 P)ATP and without sodium orthovanadate. Left panel corresponds to a representative experiment. The 32 P incorporation levels were normalized by the amount of recombinant proteins. The 32 P level of GST-Cdc14A(PD) was considered 100%. Bars show the quantification from three independent experiments. Note that incubation of Cdc14B(PD) with Cdk1 yielded increased radiolabel incorporation relative to wild-type Cdc14B acting as substrate, which demonstrates the activity of GST-Cdc14B. (d) wild-type and Cdc14A knockout RPE cells (Cdc14A −/− ) stably expressing retrovirally transduced HA-Cdc14A(PD) were treated with nocodazole during 12 hours. Rounded mitotic cells were collected (0 hr) and released into fresh medium to allow progression through mitosis. Phosphorylation state of Cdc14A(PD) was analyzed by Phos-tag gels at the indicated time points. Progression through the cell cycle was monitored by immunoblotting against the indicated proteins. C: RPE Cdc14A −/− control extracts used to identify specific Cdc14A bands. As: Asynchronous cultures. (*) Unspecific bands. Unprocessed original scans of blots and gels are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used were mouse HA-tag (12CA5, Ref. 11-666606001, Boehringer Mannheim), mouse Flag-tag (M2, peroxidase conjugated, Ref. A8592, Sigma) or Rat anti-Flag, (Ref. 299474, Agilent Technologies), mouse Myc-tag (9E10, Ref. M5546, Sigma), mouse Cdk1 (Ref. sc-54, Santa Cruz Biotechnology), rabbit phospho-Tyr15-Cdk1 (Ref. 9111, Cell Signalling), rabbit phospho-(Ser) CDKs (Ref. 2324, Cell Signaling), goat Cdc14A (N-18, Ref. sc-25952, Santa Cruz Biotechnology), rabbit Cyclin B1 (Ref. sc-752, Santa Cruz Biotechnology), mouse PP2A (ID6, Ref. 05-421, Millipore), rabbit Wee1 (Ref. sc-9037, Santa Cruz Biotechnology), mouse Plk1 (Ref. 33-1700, Zymed), rabbit phospho-histone-H3(S10) (Ref. 06-570, Millipore) and mouse β-Actin (AC-15, Ref. A5441, Sigma).

Techniques: Stable Transfection, Expressing, Phospho-proteomics, Western Blot, In Vitro, Incubation, Recombinant, Activity Assay, Knock-Out, Control

Journal: Scientific Reports

Article Title: Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

doi: 10.1038/s41598-018-30253-8

Figure Lengend Snippet: Cdc14A is phosphorylated by Cdk1 at Ser411, Ser453 and Ser549. ( a ) Schematic representation of full-length Cdc14A protein showing the distribution of all CDK phosphorylation consensus sites (S/T-P). Boxed sites represent those identified by mass spectrometric analysis of Cdc14A immunoprecipitates from HEK293T cells treated with OA. The amino-terminal and carboxy-terminal Cdc14A constructs are also indicated. (b) HEK293T cells were transfected with Flag-tagged N-terminus (ND; aa 1–332) or C-terminus (CD; aa 333–623) domains of Cdc14A. At 12 or 24 hours post-transfection, cells were treated with nocodazole (NZ, 50 ng/ml, 12 hours) or OA (0.5 μM, 2 hours), respectively. As: asynchronous, untreated cells. Cdc14A fragments were detected by immunoblotting with anti-Flag antibodies. The detection of phospho-Ser of Cdk substrates was used to confirm the efficiency of both NZ and OA treatments. Note that phosphorylation levels were higher in treated samples compared with non-treated asynchronous (As) cellular extracts. (c) Tandem mass spectrometry (MS/MS) spectra for Cdc14A phosphopeptides obtained by multistage activation CID. Representative MS/MS spectra show fragmentation of the peptides: TpSPSCAFR, MALpSPSATAK and SSNSNGGNLNpSPPGPHSAK. The peptide sequence above each representative spectrum shows theoretical “b” and “a” ion identifications (red, NH2-terminal fragments) and theoretical “y” ion identifications (blue, COOH-terminal fragments). Peaks in the spectrum that are marked red correspond to matched “b” and “a” ions, and peaks that are marked blue correspond to matched “y” ions. The number paired with each ion identification indicates the number of amino acids present. (d) HEK293T cells were transfected with Flag-Cdc14A wt, its inactive Cdc14A(PD) form or the indicated Cdc14A(PD) mutants. At 24 hours posttransfection, cells were treated with nocodazole during 12 hours and cellular extracts were obtained, resolved in Phostag-gels and analyzed by immunoblotting with anti-Flag antibodies. (e) Recombinant GST-Cdc14A(PD) and the different phosphorylation mutants (S453A, S411/549A, S411/549/453A (3A), S411/549/453/511A (4A) and S411/549/453/511/T590A (5A)) were phosphorylated in vitro in presence of γ( 32 P)ATP by Cdk1-Cyclin B1 complexes. Left panel corresponds to a representative experiment. The 32 P incorporation levels were normalized by the amount of recombinant proteins. The 32 P signal of GST-Cdc14A(PD) was considered 100%. Bars show the quantification from three independent experiments. Unprocessed original scans of blots and gels are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used were mouse HA-tag (12CA5, Ref. 11-666606001, Boehringer Mannheim), mouse Flag-tag (M2, peroxidase conjugated, Ref. A8592, Sigma) or Rat anti-Flag, (Ref. 299474, Agilent Technologies), mouse Myc-tag (9E10, Ref. M5546, Sigma), mouse Cdk1 (Ref. sc-54, Santa Cruz Biotechnology), rabbit phospho-Tyr15-Cdk1 (Ref. 9111, Cell Signalling), rabbit phospho-(Ser) CDKs (Ref. 2324, Cell Signaling), goat Cdc14A (N-18, Ref. sc-25952, Santa Cruz Biotechnology), rabbit Cyclin B1 (Ref. sc-752, Santa Cruz Biotechnology), mouse PP2A (ID6, Ref. 05-421, Millipore), rabbit Wee1 (Ref. sc-9037, Santa Cruz Biotechnology), mouse Plk1 (Ref. 33-1700, Zymed), rabbit phospho-histone-H3(S10) (Ref. 06-570, Millipore) and mouse β-Actin (AC-15, Ref. A5441, Sigma).

Techniques: Phospho-proteomics, Construct, Transfection, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy, Activation Assay, Sequencing, Recombinant, In Vitro

Journal: Scientific Reports

Article Title: Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

doi: 10.1038/s41598-018-30253-8

Figure Lengend Snippet: Cdc14A phosphorylation by Cdk1 does not inhibit its phosphatase activity. ( a , b ) HEK293T cells were co-transfected with HA-Wee1 and Flag-Cdc14A, the inactive Cdc14A(PD) form or the indicated Flag-tagged Cdc14A mutants. After 12 hours of transfection, half of the cells were treated with nocodazole during 12 hours. Then, cellular extracts were obtained from both asynchronously growing or nocodazole treated cells, and analyzed by immunoblot with the indicated antibodies. (c) GST-Cdc14A wild type (WT), GST-Cdc14A3A (3A) and GST-Cdc14A3E (3E) were purified from E . coli and processed with the PreScission protease to remove the tag. Samples were then incubated in the presence of Cdk1-Cyclin B1 complexes and cold-non-hydrolyzable ATP/γ( 32 P)ATP (100:1) for 1 hour and assayed for activity on pNPP. Reactions were performed in duplicate. Protein levels were determined by Coomassie and phosphorylation reactions by autoradiography. Right panel is representative of one of three independent experiments. The ability to hydrolyze pNPP was normalized by the amount of recombinant proteins and the phosphatase activity of non-phosphorylated GST-Cdc14Awt was considered 100%. Bars show the quantification from three independent experiments. Full-length of blots and gels are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used were mouse HA-tag (12CA5, Ref. 11-666606001, Boehringer Mannheim), mouse Flag-tag (M2, peroxidase conjugated, Ref. A8592, Sigma) or Rat anti-Flag, (Ref. 299474, Agilent Technologies), mouse Myc-tag (9E10, Ref. M5546, Sigma), mouse Cdk1 (Ref. sc-54, Santa Cruz Biotechnology), rabbit phospho-Tyr15-Cdk1 (Ref. 9111, Cell Signalling), rabbit phospho-(Ser) CDKs (Ref. 2324, Cell Signaling), goat Cdc14A (N-18, Ref. sc-25952, Santa Cruz Biotechnology), rabbit Cyclin B1 (Ref. sc-752, Santa Cruz Biotechnology), mouse PP2A (ID6, Ref. 05-421, Millipore), rabbit Wee1 (Ref. sc-9037, Santa Cruz Biotechnology), mouse Plk1 (Ref. 33-1700, Zymed), rabbit phospho-histone-H3(S10) (Ref. 06-570, Millipore) and mouse β-Actin (AC-15, Ref. A5441, Sigma).

Techniques: Phospho-proteomics, Activity Assay, Transfection, Western Blot, Purification, Incubation, Autoradiography, Recombinant

Journal: Scientific Reports

Article Title: Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

doi: 10.1038/s41598-018-30253-8

Figure Lengend Snippet: Cdk1-mediated Cdc14A phosphorylation modulates its binding pattern during mitosis. ( a ) Flag-Cdc14A or Flag-Cdc14A3A mutant expressed in HEK293T cells were immunoprecipitated with anti-Flag antibodies from asynchronously growing or nocodazole treated cells. Immunoprecipitates were resolved on SDS-PAGE gels and stained with silver solutions. As a negative control (C), extracts from HEK293T cells transfected in parallel with an empty Flag-vector were subjected to the same immunoprecipitation protocol and conditions. Asterisks show mitotic specific bands differently bound to the WT and the 3A mutant. (b) Cellular extracts used in ( a ) were analyzed by immunoblotting with the indicated antibodies. Full-length blots are shown in Supplementary Fig. . ( c ) Table showing Cdc14A-interacting proteins identified by LC-MSMS. Co-immunoprecipitates detected by silver staining in Fig. 6a (bands 1, 2, 3 and 4) were subjected to LC-MSMS analysis. From each of the four bands, all the proteins shown were identified with at least 3 unique peptides and selected from a total of 121 proteins after removing background contaminant proteins by using CRAPome program (contaminant repository for affinity purification; https:/www.crapome.org).

Article Snippet: The primary antibodies used were mouse HA-tag (12CA5, Ref. 11-666606001, Boehringer Mannheim), mouse Flag-tag (M2, peroxidase conjugated, Ref. A8592, Sigma) or Rat anti-Flag, (Ref. 299474, Agilent Technologies), mouse Myc-tag (9E10, Ref. M5546, Sigma), mouse Cdk1 (Ref. sc-54, Santa Cruz Biotechnology), rabbit phospho-Tyr15-Cdk1 (Ref. 9111, Cell Signalling), rabbit phospho-(Ser) CDKs (Ref. 2324, Cell Signaling), goat Cdc14A (N-18, Ref. sc-25952, Santa Cruz Biotechnology), rabbit Cyclin B1 (Ref. sc-752, Santa Cruz Biotechnology), mouse PP2A (ID6, Ref. 05-421, Millipore), rabbit Wee1 (Ref. sc-9037, Santa Cruz Biotechnology), mouse Plk1 (Ref. 33-1700, Zymed), rabbit phospho-histone-H3(S10) (Ref. 06-570, Millipore) and mouse β-Actin (AC-15, Ref. A5441, Sigma).

Techniques: Phospho-proteomics, Binding Assay, Mutagenesis, Immunoprecipitation, SDS Page, Staining, Negative Control, Transfection, Plasmid Preparation, Western Blot, Silver Staining, Affinity Purification

Journal: Blood Advances

Article Title: Thrombospondin-1 is an endogenous substrate of cereblon responsible for immunomodulatory drug–induced thromboembolism

doi: 10.1182/bloodadvances.2023010080

Figure Lengend Snippet: Lenalidomide-induced changes in megakaryocyte proteins coimmunoprecipitated with cereblon. (A) Experimental design for identifying human megakaryocyte–specific proteins interacting with cereblon. Lysate mixture of human megakaryocytes differentiated from CD34 + HSPCs and 293T cells transfected with FLAG-tagged cereblon were incubated with and without 10 μM lenalidomide and subjected to immunoprecipitation with antibody against FLAG. Immunoprecipitates were analyzed by sliver staining and MS. (B) Whole-cell lysates (lanes 1-2) and FLAG-specific immunoprecipitates (lanes 3-4) were subjected to silver staining after SDS–polyacrylamide gel electrophoresis. The proteins were denatured in a nonreduced condition. (C) MS signal intensities of FLAG-specific immunoprecipitates in the presence of dimethyl sulfoxide (DMSO) or 10 μM lenalidomide. Each dot shows the yield of the top 25 proteins abundant in megakaryocyte lysate ( supplemental Figure 2 ) and DDB1 (green dot).

Article Snippet: Human megakaryocytic leukemia cell line MEG01, human monocytic cell line THP-1, transfectable human embryonic kidney cells 293T, and human umbilical vein endothelial cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Transfection, Incubation, Immunoprecipitation, Staining, Silver Staining, Polyacrylamide Gel Electrophoresis

Journal: Blood Advances

Article Title: Thrombospondin-1 is an endogenous substrate of cereblon responsible for immunomodulatory drug–induced thromboembolism

doi: 10.1182/bloodadvances.2023010080

Figure Lengend Snippet: IMiDs interfere with the direct binding of THBS-1 and cereblon. (A and B) Protein expression of THBS-1 (A) and relative mRNA expression levels of THBS-1 (B) in cereblon-depleted MEG01 cells. In panel B, data are from 3 independent experiments and expressed as means ± SEM. P values were determined by 2-tailed Student t test. (C) Immunoblot analysis for THBS-1 in cereblon-depleted MEG01 cells treated with DMSO, 10 μM lenalidomide, or 10 μM pomalidomide for 24 hours. (D) Lysate mixture of MEG01 cells and 293T cells transfected with FLAG-tagged cereblon were incubated with or without 10 μM lenalidomide and subjected to immunoprecipitation with antibody against FLAG. (E) Purified recombinant His-tagged THBS-1–loaded beads were incubated with glutathione S-transferases–tagged cereblon in the presence of pomalidomide at indicated concentration. Mixture was immunoprecipitated with antibody against His. Results are representative of 3 independent experiments (panels A, C, D, and E). (F) Homologous sequences in THBS-1 and cereblon binding site of IKZF1 are aligned. The identical residues are highlighted in red. Residues of IKZF1 composing backbone of zinc-finger structure are shown in bold characters. Arrows represent indicated interacting residues between IKZF1 and cereblon in previous report. Sequence similarity with each protein was analyzed using the protein-protein BLAST program on The National Center for Biotechnology Information. CRBN, cereblon; Cont, control; GST, glutathione S-transferases; IB, immunoblotting; IP, immunoprecipitation; Len, lenalidomide; Pom, pomalidomide; Thal, thalidomide.

Article Snippet: Human megakaryocytic leukemia cell line MEG01, human monocytic cell line THP-1, transfectable human embryonic kidney cells 293T, and human umbilical vein endothelial cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Binding Assay, Expressing, Western Blot, Transfection, Incubation, Immunoprecipitation, Purification, Recombinant, Concentration Assay, Sequencing, Control

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: Identification and characterization of circPOLA2 in NSCLC (A) Volcano plots show all 1797 differentially expressed circRNAs filtered at p ≤ 0.05 in 4 paired NSCLC tissues and matched normal tissues. (B) Venn diagram showing that 5 circRNAs were overlapped by our RNA-Seq data and two GEO datasets ( GSE101684 and GSE112214 ). (C) Schematic diagram shows the genomic location of circPOLA2 and the back-splicing site confirmed by Sanger sequencing. (D) PCR with divergent or convergent primers and agarose gel electrophoresis demonstrated the presence of circPOLA2 and linear POLA2 in A549 and PC9 cells. (E and F) The stability of circPOLA2 and linear POLA2 was tested by RT-qPCR after treatment with RNase R (E) and actinomycin D (F). (G) Relative circPOLA2 levels in different NSCLC cell lines and normal bronchial epithelial cells were measured by RT-qPCR. (H) Relative circPOLA2 levels in 51 paired NSCLC tissues and adjacent normal tissues were measured by RT-qPCR. (I) ROC curve analysis was used to evaluate the diagnostic value of circPOLA2 for NSCLC ( n = 102). (J) Nuclear-cytoplasmic fractionation and RT-qPCR assays indicated that circPOLA2 was mainly distributed in the cytoplasmic of NSCLC cells; U6 and GAPDH genes were used as nuclear and cytoplasmic controls, respectively. (K) RNA-FISH shows the cytoplasm localization of circPOLA2; the circPOLA2 probe was labeled with Cy3 (red), while nuclei were stained with DAPI (blue); scale bar: 20 μm. Data are presented as the mean ± SD ( n = 3 per group, unless otherwise indicated). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns: not significant. See also Table S4 .

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: RNA Sequencing, Sequencing, Agarose Gel Electrophoresis, Quantitative RT-PCR, Diagnostic Assay, Fractionation, Labeling, Staining

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: CircPOLA2 suppressed the proliferation and aggressiveness of NSCLC cells in vitro (A) RT-qPCR measured the relative expression of circPOLA2 and linear POLA2 in A549 and PC9 cells stably transfected with circPOLA2 overexpression or knockdown plasmid. (B) Cell growth curves were plotted by CCK-8 assays after circPOLA2 overexpression or knockdown in A549 and PC9 cells. (C and D) An EdU incorporation assay was used to evaluate proliferation; scale bar: 50 μm. (E and F) Colony formation assay was used to assess cell survival and growth ability. (G and H) Transwell assay was used to measure cell migration and invasion ability (magnification: ×100). Data are presented as the mean ± SD ( n = 3 per group, unless otherwise indicated). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Stable Transfection, Transfection, Over Expression, Knockdown, Plasmid Preparation, CCK-8 Assay, Colony Assay, Transwell Assay, Migration

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: CircPOLA2 sensitized NSCLC cells to ferroptosis in vitro and suppressed tumorigenesis in vivo (A–E) A549 and PC9 cells with or without circPOLA2 overexpression were treated with erastin (10 μmol/L or indicated concentration) or erastin plus Fer-1 (1 μmol/L), DFO (50 μmol/L), Z-VAD (10 μmol/L), 3-MA (2 mmol/L), or Nec-1 (10 μmol/L) for 20 h. (A) Intracellular ATP levels were measured after treatment with different concentrations of erastin. (B) Intracellular ATP levels were measured after treatment with erastin (10 μmol/L) or erastin plus different cell death inhibitors. (C and D) Representative flow cytometry histogram of lipid peroxides stained with BODIPY 581/591 C11 (C), and the statistical results of the mean fluorescence intensity of oxidized BODIPY 581/591 C11 (D). (E) Intracellular MDA levels were measured by colorimetry, and the results were expressed as μmol per mg protein. (F) TEM images of mitochondria from PC9 cells treated with erastin (10 μmol/L) or DMSO for 20 h. (G–I) PC9 cells with stable circPOLA2 overexpression or negative control were injected into the dorsal flanks of nude mice ( n = 5 per group); gross tumor appearance in vivo and ex vivo (G); in vivo growth curves were plotted by the tumor sizes measured weekly (H), and weight was measured at the endpoint (I). Data are presented as the mean ± SD ( n = 3 per group, unless otherwise indicated). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S1 .

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: In Vitro, In Vivo, Over Expression, Concentration Assay, Flow Cytometry, Staining, Fluorescence, Colorimetric Assay, Negative Control, Injection, Ex Vivo

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: CircPOLA2 directly interacted with Merlin (A and B) RNA pull-down assay was performed using the biotin-labeled antisense or sense (negative control) probes for the cirPOLA2 back-splicing site; the protein products were used for silver staining (A) and mass spectrometry (MS) analysis (B). (C) Venn diagram showing that 7 proteins overlapped with our MS data and ferroptosis regulators. (D and E) RIP experiments were performed in A549 cells with or without circPOLA2 overexpression using anti-Merlin antibody or IgG control; the enrichment of circPOLA2 was detected by RT-qPCR (D) or RT-PCR followed by agarose gel electrophoresis (E). (F) FISH and IF double staining shows the colocalization of circPOLA2 (red) and Merlin (green) in A549 and PC9 cells; nuclei were stained with DAPI (blue); scale bar: 20 μm. (G) Schematic diagram showing the design of the truncated Merlin (top panel); RIP assay using anti-Flag antibody was performed to validate the binding domain of Merlin with circPOLA2 (bottom panel). Data are presented as the mean ± SD ( n = 3 per group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Table S3 .

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: Pull Down Assay, Labeling, Negative Control, Silver Staining, Mass Spectrometry, Over Expression, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Double Staining, Staining, Binding Assay

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: CircPOLA2 activated the Hippo signaling pathway by inhibiting the phosphorylation of Merlin (A) Representative WB results show Hippo pathway activity in NSCLC cells with circPOLA2 overexpression or knockdown. (B) The expression levels of classical oncogenes downstream of YAP were measured by RT-qPCR. (C–F) Bidirectional rescue experiments were performed to examine the necessity of Merlin. Specifically, we silenced Merlin in circPOLA2-overexpressing PC9 cells and overexpressed a constitutively activated Merlin mutant (S518A) in circPOLA2-silenced A549 cells. Representative WB results showing Hippo pathway activity (C, D); the expression of classical oncogenes downstream of YAP was measured by RT-qPCR (E, F). Data are presented as the mean ± SD ( n = 3 per group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S2 .

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: Phospho-proteomics, Activity Assay, Over Expression, Knockdown, Expressing, Quantitative RT-PCR, Mutagenesis

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: CircPOLA2 regulated the ferroptosis sensitivity and tumorigenicity of NSCLC cells via Merlin CircPOLA2-overexpressing or empty vector was cotransfected with plasmids expressing Merlin-shRNAs or shNC into PC9 cells, and stably transfected cells were obtained for experiments. (A) Cell growth curves were plotted using CCK-8 assays. (B and C) An EdU incorporation assay was used to evaluate proliferation ability; scale bar: 50 μm. (D and E) Colony formation, migration, and invasion assays were used to assess cell growth and aggressiveness (original magnification in Transwell assays: ×100). (F) Intracellular ATP levels were measured after treatment with different concentrations of erastin for 20 h. (G–I) Cells were treated with erastin (10 μmol/L) or DMSO for 20 h; representative flow cytometry histogram of lipid peroxides stained with BODIPY 581/591 C11 (G), and the statistical results of the mean fluorescence intensity of oxidized BODIPY 581/591 C11 (H); intracellular MDA levels were measured by colorimetry, and the results were expressed as μmol per mg protein (I). (J) TEM images of mitochondria from PC9 cells treated with erastin (10 μmol/L) for 20 h. Data are presented as the mean ± SD ( n = 3 per group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figures S3 and .

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: Plasmid Preparation, Expressing, Stable Transfection, Transfection, CCK-8 Assay, Migration, Flow Cytometry, Staining, Fluorescence, Colorimetric Assay

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet: CircPOLA2 sensitized NSCLC cells to ferroptosis and inhibited tumorigenesis via Merlin in vivo The xenograft model was established by injecting PC9 cells stably overexpressing circPOLA2 with or without Merlin-shRNAs into the flanks of BALB/c nude mice. (A) Gross tumor appearance in vivo and ex vivo . (B) Growth curves were plotted from tumor sizes measured weekly. (C) Tumor weight was measured at the endpoint. (D and E) Representative pictures of Ki-67 stained by IHC (D) and statistical analysis of the staining intensity score (E). (F and G) Representative pictures of 4-HNE stained by IHC (F) and statistical analysis of the staining intensity score (G). (H) MDA levels in tumor lysates were measured by colorimetry, and the results were expressed as μmol per mg protein. (I) Schematic illustrating the role of circPOLA2 in NSCLC. Data are presented as the mean ± SD ( n = 5 per group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S5 .

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: In Vivo, Stable Transfection, Ex Vivo, Staining, Colorimetric Assay

Journal: iScience

Article Title: CircPOLA2 sensitizes non-small cell lung cancer cells to ferroptosis and suppresses tumorigenesis via the Merlin-YAP signaling pathway

doi: 10.1016/j.isci.2024.110832

Figure Lengend Snippet:

Article Snippet: The human NSCLC cell lines A549, H1975, and H1299 and the human normal bronchial epithelial cell line BBM were acquired from the American Type Culture Collection (ATCC, USA).

Techniques: Control, Virus, Recombinant, Magnetic Beads, Protease Inhibitor, Silver Staining, Multiple Displacement Amplification, Purification, Cell Viability Assay, CCK-8 Assay, In Vitro, Sequencing, Expressing, Plasmid Preparation, Cloning, Software

Journal: Experimental & Molecular Medicine

Article Title: Protective effect of hepatocyte-enriched lncRNA- Mir122hg by promoting hepatocyte proliferation in acute liver injury

doi: 10.1038/s12276-022-00881-2

Figure Lengend Snippet: a , b For the overexpression experiment in vivo, mice were injected with AAV8-Mir122hg or control AAV8-TBG for 14 d, followed by CCl 4 or oil treatment for 24 h ( n = 8 per group). For the silencing experiment in vivo, mice were injected with AAV8-sh-Mir122hg or AAV8-sh-control for 14 d, followed by CCl 4 or oil treatment for 48 h ( n = 8 per group). The levels of AKT signaling-related p-AKT (Ser473), p-AKT (Thr308), p-GSK-3β, cell cycle-inhibitory p27, p-p27, and the cell cycle-related proteins cyclin E and CDK2 in each group were detected by WB a , b . c – f For the overexpression in vitro, primary HCs were treated with lentivirus LV-Mir122hg or LV-Control for 48 h, followed by TNFα treatment for 24 h; for the silencing experiment in vitro, primary HCs were treated with si-Mir122hg or control si-NC for 36 h, followed by TNFα treatment for 24 h; cellular p-AKT (ser473), p-AKT (Thr308), p-GSK-3β, p27, p-p27, cyclin E, CDK2 levels were detected by WB c , e ; p-AKT (ser473) and p27 levels were also detected by confocal microscopy d , f . g – i Primary HCs were treated with lentivirus LV-Mir122hg or LV-Control for 48 h, followed by treatment with TNFα and AKT specific blocker MK2206 or control DMSO for 24 h; Cellular RNA levels of Mir122hg-001 , Pcna , Ki67 were measured by qPCR g ; protein levels of p-AKT(ser473), p-AKT(thr308), p-GSK-3β, p-c-Raf, mTOR, p-mTOR h , and downstream proteins LC3B, p27, p-p27, Cyclin E, CDK2, and PCNA were detected by WB i . The data are shown as the mean ± SD of at least triplicate experiments. * p < 0.05 vs. Control, # p < 0.05 vs. LV-Mir122hg+TNFα + DMSO.

Article Snippet: The cells were treated with 4% paraformaldehyde overnight at 4 °C, permeabilized in 1% Triton X-100 for 0–60 min, and blocked with 10% goat serum for 30 min at room temperature. p-AKT (Ser473) (Cell Signaling, 4060) and p27 (Abcam, ab32034) were incubated overnight at 4 °C, and unrelated isotype rabbit IgG (Millipore, PP64B) was used as a negative control.

Techniques: Over Expression, In Vivo, Injection, Control, In Vitro, Confocal Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Protective effect of hepatocyte-enriched lncRNA- Mir122hg by promoting hepatocyte proliferation in acute liver injury

doi: 10.1038/s12276-022-00881-2

Figure Lengend Snippet: a Under the condition of |correlation coefficient R | > 0.5 and p < 0.05, genes that were closely associated with Mir122hg were screened in the three RNA-seq datasets of APAP-, hepatectomy-, and bacterial infection-induced liver injury, and further GO and KEGG analyses were performed by R software with the clusterProfiler package. b , c For the overexpression in vitro, primary HCs were treated with lentivirus LV-Mir122 hg or LV-Control for 48 h, followed by TNFα treatment for 24 h; for the silencing experiment in vitro, primary HCs were treated with si-Mir122 hg or control si-NC for 36 h, followed by TNFα treatment for 24 h; mRNA levels of Cxcr1, Cxcr2, Cxcl3, Cxcl5, Cxcl7, and Cxcl8 were measured by qPCR. d , f Primary HCs were treated with lentivirus LV-Mir122hg or LV-Control for 12 h, followed by treatment with CXCR2 specific antagonist SB225002 or control DMSO for 48 h; cellular RNA levels of Mir122hg-001 , Pcna , Ki67 were measured by qPCR d , protein levels of p-AKT(ser473), p-AKT(thr308), p-GSK-3β, p-c-Raf, mTOR, p-mTOR e , and p27, p-p27(s10), Cyclin E, CDK2 and PCNA were detected by WB f . The data are shown as the mean ± SD of at least triplicate experiments. * p < 0.05 vs. Control, # p < 0.05 vs. LV-Control+TNFα or si-NC + TNFα or LV-Mir122hg+ DMSO.

Article Snippet: The cells were treated with 4% paraformaldehyde overnight at 4 °C, permeabilized in 1% Triton X-100 for 0–60 min, and blocked with 10% goat serum for 30 min at room temperature. p-AKT (Ser473) (Cell Signaling, 4060) and p27 (Abcam, ab32034) were incubated overnight at 4 °C, and unrelated isotype rabbit IgG (Millipore, PP64B) was used as a negative control.

Techniques: RNA Sequencing, Infection, Software, Over Expression, In Vitro, Control

Journal: Experimental & Molecular Medicine

Article Title: Protective effect of hepatocyte-enriched lncRNA- Mir122hg by promoting hepatocyte proliferation in acute liver injury

doi: 10.1038/s12276-022-00881-2

Figure Lengend Snippet: a Sense and antisense strand RNA of Mir122hg-001 was obtained by in vitro transcription, and RNA‒protein pulldown experiments were performed with biotin magnetic beads; the protein samples obtained were subjected to silver staining, and mass spectrometry revealed 11 possible binding proteins. b , c qPCR and PCR (including agarose gel electrophoresis) detection of Mir122hg-001 retrieved by anti-C/EBPα-specific antibody compared with Control IgG in the RIP assay with primary HCs. d Primary HCs were infected with LV-Control or LV-Mir122hg, ChIP analyses were performed on the indicated gene promoter regions using an anti-C/EBPα-specific antibody, and enrichment is shown relative to the input. e , f Primary HCs were treated with si-Mir122hg or si-Cebpa or control si-NC for 36 h. Cellular RNA levels of Cebpa , Pcna , Ki67 , Cxcr1 , Cxcr2 , Cxcl1 , Cxcl2 , Cxcl3 , Cxcl5 , Cxcl7 and Cxcl8 were measured by qPCR e . Protein levels of C/EBPα, p-AKT (ser473), p-AKT (thr308), p-GSK-3β, p-c-Raf, p27, p-p27 (s10), Cyclin E, CDK2 and PCNA were detected by WB f . The data are shown as the mean ± SD of at least triplicate experiments. * p < 0.05 vs. Control or si-NC.

Article Snippet: The cells were treated with 4% paraformaldehyde overnight at 4 °C, permeabilized in 1% Triton X-100 for 0–60 min, and blocked with 10% goat serum for 30 min at room temperature. p-AKT (Ser473) (Cell Signaling, 4060) and p27 (Abcam, ab32034) were incubated overnight at 4 °C, and unrelated isotype rabbit IgG (Millipore, PP64B) was used as a negative control.

Techniques: In Vitro, Magnetic Beads, Silver Staining, Mass Spectrometry, Binding Assay, Agarose Gel Electrophoresis, Control, Infection

Journal: Genes

Article Title: Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells

doi: 10.3390/genes12020264

Figure Lengend Snippet: Cytotoxic effect of bisphenol A (BPA) on Y79 retinoblastoma cells. Cells were treated with the indicated concentration of BPA for 48 h. ( a ) Morphological changes in Y79 cells were observed by phase-contrast microscopy after BPA treatment. Cell viability was analyzed using the live/dead assay. Fluorescence microscopy images of live or dead cells after 30 min staining with calcein-AM (0.3 μM, Live cells) and EthD-1 (3 μM, Dead cells). Scale bars = 200 μm. ( b ) Cellular behavior by BPA treatment measured using FACS analysis. Cells were stained antibodies against each markers; Ki-67 for cell proliferation, propidium iodide (PI) for cell cycle. ( c ) Cyclin B1 and cyclin D1 for detection of cell cycle markers, Annexin-V/PI staining for apoptosis, cleaved Caspase-3 for detection of the active apoptosis. Values are presented as mean ± S.E.M., n = 6.

Article Snippet: Human retinoblastoma Y79 cells were purchased from the ATCC (Manassas, VA, USA).

Techniques: Concentration Assay, Microscopy, Live Dead Assay, Fluorescence, Staining

Journal: Genes

Article Title: Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells

doi: 10.3390/genes12020264

Figure Lengend Snippet: Dynamic transcriptome profiles by the BPA treatment in Y79 retinoblastoma cells. Comparative analysis of transcriptome generated by directional RNA-seq from BPA treated Y79 cells. ( a ) Principal component analysis (PCA) of directional RNA-seq data. The values reveal the amount of variation attributed to each principal component. Small circles indicate individual samples, and larger ones show each group. ( b ) Transcriptional pattern analysis in control and BPA-exposed groups by employing heat map and hierarchical clustering. ( c ) Volcano plot shows differentially expressed transcripts in BPA treated group compared to the DMSO-treated group. Small red circled transcripts were verified as having the absolute value of log2 (fold changes) and −log10 ( p -value). ( d ) Differentially expressed transcripts were identified by the DEseq in BPA treated group from total annotated transcripts. ( e ) Heatmap depicting fold changes for all transcripts indicating statistically significant differences between the BPA-treated group and the control group.

Article Snippet: Human retinoblastoma Y79 cells were purchased from the ATCC (Manassas, VA, USA).

Techniques: Generated, RNA Sequencing, Control

Journal: Genes

Article Title: Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells

doi: 10.3390/genes12020264

Figure Lengend Snippet: Analysis of gene ontology in cells treated with BPA. The classification of gene ontology (GO) functional enrichment analyses with the differentially expressed genes (DEGs) from comparisons of control and BPA-treated Y79 cells. The functional enriched classes of downregulated DEGs annotated by biological process ( a ) and molecular function ( b ).

Article Snippet: Human retinoblastoma Y79 cells were purchased from the ATCC (Manassas, VA, USA).

Techniques: Functional Assay, Control

Journal: Genes

Article Title: Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells

doi: 10.3390/genes12020264

Figure Lengend Snippet: Transcriptome dynamics identifies the alternative splicing events in BPA-treated Y79 cells. ( a ) Alternative splicing events were analyzed by the MATS and identifies alternative splicing events. Types of alternative splicing events detected and counted their frequencies in group-wise comparisons. Protein coding genes obtained from alternative splicing events indicated as the percentage of genes. Differential splicing events in the BPA-treated group shown the clear retained intron (RI) in Mago Homolog B, Exon Junction Complex Subunit ( MAGOHB ) ( b ) and Heterogeneous Nuclear Ribonucleoprotein D ( HNRNPD ) ( c ) genes. The yellow shadow indicates the retained intron region in BPA-treated Y79 cells.

Article Snippet: Human retinoblastoma Y79 cells were purchased from the ATCC (Manassas, VA, USA).

Techniques: Alternative Splicing

Journal: Genes

Article Title: Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells

doi: 10.3390/genes12020264

Figure Lengend Snippet: Comparative proteome analysis by low-dose of BPA in Y79 cells. ( A ) Representative silver staining images of 2D electrophoresis patterns from BPA-treated and control groups in Y79 cells. Each spots indicate differentially expressed proteins (red arrow), up- and downregulated proteins, respectively. HIST2H4B (1), PDIA5 (2), HYOU1 (3), VCP (4), PARK7 (5), TBP (6), GAPDH (7), MTHFD2 (8), LDHA (9). ( B ) The nine proteins identified that significant up- and downregulated by exposure to BPA. ( C ) Selected proteins are represented as fold changes (log 2 ). ( D ) The protein network was identified using the STRING database. Network nodes represent proteins shown by gene names and colored lines represent protein–protein interactions. ( E ) Western blot analysis of HYOU1. β-actin was used as a protein loading control.

Article Snippet: Human retinoblastoma Y79 cells were purchased from the ATCC (Manassas, VA, USA).

Techniques: Silver Staining, Two-Dimensional Gel Electrophoresis, Control, Protein-Protein interactions, Western Blot

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: Rs1883832 is the fSNP of CD40 . ( A ) Luciferase reporter assay showed that among the three candidate SNPs that scored 5, only rs1883832 had allele-imbalanced luciferase activity. The luciferase reporter with SNP-centered fragment containing either the risk or non-risk allele was transfected into HepG2 cells. Relative luciferase activity was analyzed between the risk (teal) and non-risk (orange) alleles of the three candidate SNPs. ( B ) EMSA showed, among the three candidate SNPs that scored 5, only rs1883832 had allele-imbalanced binding activity of nuclear protein. The 31-bp biotinylated DNA fragments centered on each SNP were established and incubated with nuclear extract from HepG2 cells. The binding activity of nuclear protein in risk allele and non-risk allele was evaluated. rs1883832: T, risk allele; C, non-risk allele; rs6074022: C, risk allele; T, non-risk allele; rs4810485: T, risk allele; G, non-risk allele. Red arrow, allele-specific shift. Lane 1, risk allele probe only; lane 2, non-risk allele probe only; lane 3, risk allele probe with nuclear extract; lane 4, non-risk allele probe with nuclear extract. ( C ) WashU Epigenome Browser showed H3K4me3 (the signals of promoter-associated chromatin modification) enrichment tracks at the rs1883832 locus in HepG2 cells and liver, H3K27ac (the signals of enhancer-specific chromatin modification) enrichment tracks of HepG2, along with DNAse signals (the probability of the existence of enhancer activity) in HepG2, hepatocyte and liver. Experiments were performed in triplicate. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Incubation, Modification

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: ANXA2 is the regulatory protein which preferentially binds to the risk allele T of rs1883832. ( A ) The schematic of FREP was diagrammed. Briefly, the FREP constructed with BamHI (cyan) and EcoRI (green) restriction sites flanking a 31-bp sequence (light blue) centered on the fSNP of interest (red), and two 20-bp sequences (orange) for PCR were attached to magnetic dynabeads. After incubation of nuclear extract, EcoRI digestion deleted the 3′ DNA binding proteins followed by removal of the 5′ non-specific DNA complex by BamHI digestion. With magnetic separation and wash, the remaining fraction was analyzed by mass spectrometry (MS). ( B ) MS discovered that several proteins may bind with the rs1883832 region. The silver stain (left) showed proteins pulled down from lane 1 (non-specific sequence) and lane 2 (test sequence with rs1883832). The red arrow indicated the specific bands sent for MS analysis. The peptide spectrum count was listed (right). ( C ) ANXA2 regulated CD40 expression. Western blot was performed to assess the expression of CD40 after siRNA-mediated silencing of ANXA2. ( D ) ChIP-qPCR (left) and ChIP-PCR (right) were conducted to investigate endogenous binding of ANXA2 to the rs1883832 region. The agarose gel electrophoresis of PCR products for Input and ChIP, respectively. Light blue arrows indicated the marker bands. The enrichment with ANXA2-specific antibody compared with IgG antibody, as well as between negative control (siCtrl) and ANXA2-si3 HepG2 cells, was evaluated. ( E and F ) ChIP experiment combined with TA cloning demonstrated that ANXA2 preferentially bound to the risk allele T of rs1883832. (E) A sequencing trace was performed to explore the genotypes on rs1883832 in hepatocarcinoma cell lines. Rs1883832 DNA fragments from both input and ChIP samples were cloned into TA vectors. (F) The number of clones containing allele T or C in each group was counted. Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Construct, Sequencing, Incubation, DNA Binding Assay, Mass Spectrometry, Silver Staining, Expressing, Western Blot, ChIP-qPCR, Binding Assay, Agarose Gel Electrophoresis, Marker, Negative Control, TA Cloning, Clone Assay

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: CD40 exerts anti-HBV effect in HBV-producing cells, HBV-transfected and HBV-infected cells. ( A – C ) Inhibition of CD40 or CD40L using antagonistic antibodies, or knockdown of CD40 via siRNA reversed suppression of HBV replication and transcription. HepG2.2.15 cells transfected with CD40 siRNA or siCtrl were stimulated with CD40L (1 μg/ml) by preincubation with anti-CD40 antibody (5 μg/ml) or anti-CD40L antibody (0.1 μg/ml) for 1 h. The level of extracellular HBV DNA load was determined using the Hepatitis B Viral DNA Quantitative Fluorescence Diagnostic Kit (A). The expression of intracellular HBsAg and HBcAg was analyzed by western blot (B). HepG2.2.15 cells were transfected with CD40 siRNA or siCtrl for 72 h and the expression of intracellular HBV total RNA and HBV 3.5-kb RNA was measured by qPCR (C). ( D – E ) Decreased expression of CD40 facilitates HBV transcription. HBV-transfected HepG2 cells were transfected with CD40 siRNA or siCtrl for 72 h. HepG2-NTCP cells were infected with 1000 genome equivalents/cell of HBV for 24 h and then transfected with CD40 siRNA or siCtrl for 96 h. Expression of intracellular HBV total RNA, 3.5-kb RNA, HBsAg and HBcAg was analyzed as indicated (D and E). ( F – H ) Overexpression of CD40 resulted in the suppression of HBV replication and transcription. HepAD38 cells were transfected with CD40 plasmid or vector for 72 h and HBV DNA load in cell supernatant was determined as indicated (F). HBV-transfected HepG2 cells were transfected with CD40 plasmid or vector for 72 h. HepG2-NTCP cells were infected with 1000 genome equivalents/cell of HBV for 24 h and then transfected with CD40 plasmid or vector for 96 h. Expression of intracellular HBV total RNA, 3.5-kb RNA, HBsAg and HBcAg was analyzed as indicated (G and H). Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Transfection, Infection, Inhibition, Knockdown, Fluorescence, Diagnostic Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: CD40 activates JAK–STAT signaling pathway. ( A – B ) Total RNA from HepG2.2.15 transfected with CD40 siRNA or siCtrl was extracted and used for mRNA sequencing analysis. GO enrichment analysis (A) and KEGG enrichment analysis (B) were performed by hypergeometric test. P value was adjusted by the false discovery rate (FDR) method. ( C ) The expression of STAT1, p-STAT1, STAT3, p-STAT3 and IRF9 was detected in CD40-silenced cells, and CD40-overexpressed cells with or without preincubation of STATs inhibitor (nifuroxazide 20 μM) for 24 h. ( D ) The level of IRF9 in nucleus and cytoplasm was examined in HBV-transfected HepG2 and Huh7 cells. ( E ) Immunofluorescence assay was utilized to observe the subcellular location of IRF9. Red for IRF9, blue for DAPI. ( F ) Luciferase activity was analyzed after ISRE reporter plasmids co-transfection into cells with CD40 siRNA/siCtrl or CD40 plasmid/vector for 48 h. Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Transfection, Sequencing, Expressing, Immunofluorescence, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: CD40 induces BST2 expression. ( A – B ) BST2 was selected as downstream target gene of JAK–STAT pathway. Several JAK–STAT pathway related genes in HepG2.2.15 (A) and HepAD38 (B) transfected with CD40 siRNA were detected by qPCR. ( C – E ) CD40 modulated BST2 expression via JAK–STAT pathway. In HBV-transfected and HBV-infected model, the level of BST2 was detected in CD40-silenced cells, and CD40-overexpressed cells with or without preincubation of STATs inhibitor (nifuroxazide 20 μM) for 24 h (C). HBV-transfected HepG2 and Huh7 cells transfected with CD40 siRNA or siCtrl were seeded in 24-well plate (2 × 10 4 cells/well) and cultured for 48 h, and then the expression of BST2 was analyzed by immunofluorescence assay (D and E). Red for BST2, blue for DAPI. ( F – G ) CD40 is positively associated with BST2. Correlation of CD40 and BST2 in liver tissues was plotted by the GTEx database (F). Correlation of CD40 and BST2 in HBV infected liver tissues from published data (GSE84044) was plotted (G). Experiments were performed in triplicate.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Expressing, Transfection, Infection, Cell Culture, Immunofluorescence

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: Suppression of JAK/STAT/BST2 axis abrogates CD40-induced inhibitory effect of HBV. ( A – C ) Overexpression of BST2 restrained HBV replication and transcription. HBV DNA load in HepAD38 cells supernatant was determined. Intracellular HBV total RNA, HBV 3.5-kb RNA, HBsAg and HBcAg in HBV-transfected HepG2, or HBV-infected HepG2-NTCP cells transfected with BST2 plasmid or vector were analyzed as indicated. ( D – E ) Knockdown of BST2 partially abrogated overexpressed CD40-induced suppression of HBV replication and transcription. CD40 plasmid and BST2 siRNA were sequentially transfected into HBV-transfected Huh7 and HBV-infected HepG2-NTCP cells, and then intracellular HBV total RNA, HBV 3.5-kb RNA, HBsAg and HBcAg were detected. ( F – G ) Inhibition of the JAK/STAT pathway with nifuroxazide decreased BST2 expression, and restored HBV replication and transcription which were suppressed by CD40. HBV-infected HepG2-NTCP cells and PHHs were transfected with CD40 plasmid for four days and then treated with nifuroxazide for one more day. Expression of intracellular HBV total RNA, HBV 3.5-kb RNA, HBsAg and HBcAg was analyzed. Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Over Expression, Transfection, Infection, Plasmid Preparation, Knockdown, Inhibition, Expressing

Journal: Advanced Science

Article Title: SenExo‐cCCT2 Reprograms Senescence Response and Anti‐Tumor Immunity Following FOLFIRINOX Chemotherapy in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202508431

Figure Lengend Snippet: Correlation of tumor cell senescence with chemosensitivity and CD8 + T‐cell infiltration in PDAC A) The overall experimental scheme of this study. B) t‐SNE plots of the four tumor cell (TC) clusters sorted from the human PDAC scRNA‐seq data. C) Dot plot of senescence marker genes of the four tumor cell (TC) clusters, with the dot size representing the percent expression in the cluster and the dot color scaled by the expression level. D) t‐SNE plots showing the expression of senescence marker genes. E) GSVA showing the average GSVA score per cluster for senescence‐related pathways. Database: Fridman (F), Hallmark (H), Reactome (R). F) Comparison of the senescence‐apoptosis ratio (SAR) across chemotherapy regimens. G) Representative MRI and mIF images for FOLFIRINOX resistant (R) and sensitive (S) patients, demonstrating PDAC size, senescence and apoptosis distribution, and CD8 + T‐cell infiltration after chemotherapy. Orange circles: primary pancreatic tumors. Scale bar, 50 µm. H) Comparison of the SAR by response status within the FOLFIRINOX subgroup. I) t‐SNE plots of the seven identified T lymphocyte clusters sorted from the human PDAC scRNA‐seq data. J) The proportions of different defined T lymphocyte types in the T lymphocyte population. K) Network Venn diagram illustrating the overlap between differentially expressed circRNAs between the FOLFIRINOX‐R and FOLFIRINOX‐S groups and the senescence high‐ and low‐ groups. L) Dot plot demonstrating the correlation between the seven circRNAs screened and senescence‐related pathways, with dot size and dot color representing the correlation coefficient. M) Heat map depicting the percentage of SA‑β‑Gal‑positive cells following overexpression or knockdown of the seven candidate circRNAs. N) Heat map depicting relative cell viability following overexpression or knockdown of the seven candidate circRNAs. O) Representative images of H&E staining and cCCT2 immunohistochemistry in the FOLFIRINOX‐R and FOLFIRINOX‐S PDAC tissues and patient‐derived organoid (PDO). Data are expressed as the means ± SDs. Unpaired two‐tailed Student's t‐test or Mann–Whitney U ‐test (F, H), Spearman analysis (L). Not significant (ns); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Figure created with BioRender.com.

Article Snippet: The human PDAC cell lines PANC‐1 (RRID: CVCL_0480), BxPC‐3 (RRID: CVCL_0186), Capan‐1 (RRID: CVCL_0237), MIA PaCa‐2 (RRID: CVCL_0428), AsPC‐1 (RRID: CVCL_0152) and SW1990 (RRID: CVCL_1723) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Marker, Expressing, Comparison, Over Expression, Knockdown, Staining, Immunohistochemistry, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Advanced Science

Article Title: SenExo‐cCCT2 Reprograms Senescence Response and Anti‐Tumor Immunity Following FOLFIRINOX Chemotherapy in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202508431

Figure Lengend Snippet: cCCT2 overexpression mediates cellular senescence following FOLFIRINOX in PDAC A) Schematic structure of cCCT2 and Sanger sequencing analysis of the back splicing junction in endogenously amplified cCCT2. B) RNA stability analysis of cCCT2, CCT2, and GAPDH in RNase R‐treated Capan‐1 cells. GAPDH mRNA was used as a negative control (n = 3 per group). C) RT–qPCR analysis of cCCT2 and CCT2 stability in Capan‐1 cells following actinomycin D treatment (n = 3 per group). GAPDH mRNA serves as negative control. D–H) Bar chart showing cell viability of EV‑ and OE‑cCCT2‑expressing Capan‐1 cells after treatment with the active metabolite of irinotecan SN38 (D), oxaliplatin (E), Fluorouracil (F), Gemcitabine (G), and three‐drug combination FOI (H) at the indicated concentrations (n = 6 per group). Bars show the ​log‐transformed viability ratio​ of cCCT2‐overexpressing cells relative to vector controls. Data display using mean with range. Bars extending below the baseline (0) indicate a reduction in viability of OE‑cCCT2 cells relative to EV controls. * P < 0.05. I) Representative images of the proliferation of Capan‐1 and MIA PaCa‐2 3D microtumor spheroids with cCCT2 overexpression or knockdown and quantification of the results (n = 6 per group). Scale bar, 100 µm. J) Representative images of the apoptosis of Capan‐1 and MIA PaCa‐2 3D microtumor spheroids following cCCT2 overexpression or knockdown and quantification of the results (n = 6 per group). Live cells were stained with calcein‐AM (green), and dead cells were stained with propidium iodide (PI) (red). Scale bar, 100 µm. K) Western blot analysis of apoptosis‑related protein expression in Capan‑1 cells overexpressing cCCT2 (n = 3). L) Representative images of SA‐β‐Gal‐staining in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 30 µm. M) Western blot analysis of senescence‑related protein expression in Capan‑1 cells overexpressing cCCT2 (n = 3). N) Tumor volume growth curves and weights for xenograft nude mouse models generated with cCCT2‐overexpressing Capan‐1 and cCCT2‐knockdown MIA PaCa‐2 cells (n = 6 per group). O) Representative images of H&E staining, IHC staining for caspase‐3, and SA‐β‐Gal staining of tumor tissues from mice with cCCT2 overexpression or knockdown. Scale bar, 100 µm. Data are expressed as the means ± SDs. Unpaired two‐tailed Student's t‐test or Mann–Whitney U ‐test (B), paired t‐test or Wilcoxon signed‐rank test (D–H), repeated measures ANOVA test (C, N), two‐way ANOVA with Tukey's post‐hoc test (I, J, L). Not significant (ns); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The human PDAC cell lines PANC‐1 (RRID: CVCL_0480), BxPC‐3 (RRID: CVCL_0186), Capan‐1 (RRID: CVCL_0237), MIA PaCa‐2 (RRID: CVCL_0428), AsPC‐1 (RRID: CVCL_0152) and SW1990 (RRID: CVCL_1723) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Sequencing, Amplification, Negative Control, Quantitative RT-PCR, Transformation Assay, Plasmid Preparation, Knockdown, Staining, Western Blot, Expressing, Generated, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Advanced Science

Article Title: SenExo‐cCCT2 Reprograms Senescence Response and Anti‐Tumor Immunity Following FOLFIRINOX Chemotherapy in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202508431

Figure Lengend Snippet: cCCT2 overexpression enhances FOLFIRINOX‐induced cellular senescence via DNA damage accumulation A) Representative images of 8‐OHdG accumulation in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 30 µm. B) Representative images of γ‐H2AX accumulation in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 30 µm. C) Comet assay detects DNA strand breaks in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 10 µm. Two‐way ANOVA with Tukey's post‐hoc test (A–C). Not significant (ns); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The human PDAC cell lines PANC‐1 (RRID: CVCL_0480), BxPC‐3 (RRID: CVCL_0186), Capan‐1 (RRID: CVCL_0237), MIA PaCa‐2 (RRID: CVCL_0428), AsPC‐1 (RRID: CVCL_0152) and SW1990 (RRID: CVCL_1723) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Single Cell Gel Electrophoresis

Journal: Advanced Science

Article Title: SenExo‐cCCT2 Reprograms Senescence Response and Anti‐Tumor Immunity Following FOLFIRINOX Chemotherapy in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202508431

Figure Lengend Snippet: cCCT2 overexpression potentiates DNA damage accumulation via suppression of SLX4‐mediated DNA damage repair response A) Volcano plot of differentially expressed proteins in Capan‐1 with EV or overexpressing cCCT2 after FOI treatment. The dot color indicates protein expression after normalization, and the dot size represents the P value. B) Enrichment circle plot of the proteomic data showing the enrichment of senescence‐ and DDR‐related pathways. C) GSEA plot showing enrichment of the DNA damage/telomere stress‐induced senescence pathway in the Capan‐1 group with high cCCT2 expression levels. D) Heatmap of the differential expression of 276 DDR‐related proteins identified via proteomics. E) Representative images of SLX4 condensate formation in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 20 µm. F) Quantification of the effect of altering SLX4 expression levels on SLX4 condensate formation in PDAC cells with different cCCT2 expression levels (n = 6 per group). G) SA‐β‐Gal staining showing the effect of altering SLX4 expression in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 30 µm. H) Representative images of the effect of altering SLX4 expression levels on γ‐H2AX accumulation in Capan‐1 cells with EV or overexpressing cCCT2 and quantification of the results (n = 6 per group). Scale bar, 30 µm. I) Representative images of the effect of altering SLX4 expression levels on tail moment of comet assay in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 20 µm. J) Representative images of the effect of altering SLX4 expression levels on TOP1‐DPCs staining in PDAC cells with different cCCT2 expression levels and quantification of the results (n = 6 per group). Scale bar, 20 µm. Unpaired two‐tailed Student's t‐test or Mann–Whitney U ‐test (E). one‐way ANOVA with Tukey's post‐hoc test (F–J). Not significant (ns); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The human PDAC cell lines PANC‐1 (RRID: CVCL_0480), BxPC‐3 (RRID: CVCL_0186), Capan‐1 (RRID: CVCL_0237), MIA PaCa‐2 (RRID: CVCL_0428), AsPC‐1 (RRID: CVCL_0152) and SW1990 (RRID: CVCL_1723) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Quantitative Proteomics, Staining, Single Cell Gel Electrophoresis, Two Tailed Test, MANN-WHITNEY

Journal: Advanced Science

Article Title: SenExo‐cCCT2 Reprograms Senescence Response and Anti‐Tumor Immunity Following FOLFIRINOX Chemotherapy in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202508431

Figure Lengend Snippet: cCCT2 overexpression modulates SUMOylation of SLX4 via competitive binding to IPO13 A) Identification of RBP binding to specific biotin‐labeled cCCT2 (sense) and control (antisense) probes via silver staining and mass spectrometry. Labeled region of the strip corresponding to IPO13 (97 kDa). B) Interaction between cCCT2 and the IPO13 protein was detected in Capan‐1 cells via a specific biotin‐labeled cCCT2 (sense) RNA pull‐down assay. C) RT‐qPCR was performed after the RIP experiments to detect the amount of cCCT2 bound to the IPO13 protein (n = 3). D) Colocalization of cCCT2 (red) and IPO13 (green) was determined via RNA‐FISH and immunofluorescence staining, and the cell nuclei were stained with DAPI (blue). Scale bar, 10 µm. E) Colocalization of UBC9 (green) and IPO13 (red) in PDAC cells with different cCCT2 expression levels and quantification of the results. Scale bar, 30 µm. F) Assessment of nuclear and cytoplasmic distribution of UBC9 in Capan‑1 cells with EV or overexpressing cCCT2 following altering IPO13 expression levels. H3 was used as a nuclear marker, and GAPDH was used as a cytoplasmic marker. G) Measurement of SLX4 protein expression and SUMOylation levels in Capan‑1 cells with EV or overexpressing cCCT2 following the overexpression of IPO13 or UBC9. H) Assessment of the effect of altering IPO13 expression levels on SLX4 condensation formation in Capan‑1 cells with EV or overexpressing cCCT2 (n = 6 per group). I) Prediction of the structural regions on cCCT2 that can bind to IPO13 via the bioinformatics tools RNAfold and catRAPID. J) Design of mutations that disrupt the candidate binding site of cCCT2 to IPO13. K) RNA pull‐down assay to assess the binding between three cCCT2 mutants and IPO13 protein. L) RT‒qPCR after the RIP assay to assess the binding of three cCCT2 mutants and IPO13 protein (n = 3 per group). M) Assessment of the effect of cCCT2‐Mut 3 on the nuclear and cytoplasmic distribution of UBC9 in Capan‐1 cells (n = 6 per group). N) Assessment of the effect of cCCT2‐Mut 3 on SLX4 condensation formation in Capan‐1 cells (n = 6 per group). O) Assessment of the effect of cCCT2‐Mut 3 on TOP1‑DPCs accumulation in Capan‐1 cells (n = 6 per group). P) Assessment of the effect of cCCT2‐Mut 3 on apoptosis of Capan‐1 3D microtumor spheroids (n = 6 per group). Q) Assessment of the effect of cCCT2‐Mut 3 on SA‐β‐Gal staining in Capan‐1 cells (n = 6 per group). Unpaired two‐tailed Student's t‐test or Mann–Whitney U ‐test (C, L), one‐way ANOVA with Dunnett's post‐hoc test (M–Q), one‐way ANOVA with Tukey's post‐hoc test (H). Not significant (ns); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The human PDAC cell lines PANC‐1 (RRID: CVCL_0480), BxPC‐3 (RRID: CVCL_0186), Capan‐1 (RRID: CVCL_0237), MIA PaCa‐2 (RRID: CVCL_0428), AsPC‐1 (RRID: CVCL_0152) and SW1990 (RRID: CVCL_1723) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Binding Assay, Labeling, Control, Silver Staining, Mass Spectrometry, Stripping Membranes, Pull Down Assay, Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Marker, Two Tailed Test, MANN-WHITNEY

Journal: Advanced Science

Article Title: SenExo‐cCCT2 Reprograms Senescence Response and Anti‐Tumor Immunity Following FOLFIRINOX Chemotherapy in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202508431

Figure Lengend Snippet: SenExo‐cCCT2 synergizes with chemotherapy and sequential anti‐PD‐L1 therapy to suppress pancreatic cancer progression A) Schematic diagram of the exosome‐based delivery system used to construct SenExo‐cCCT2. B) Representative transmission electron microscopy images of SenExo‐cCCT2. Scale bar, 100 nm. C) Size distribution of the SenExo‐cCCT2 analyzed with a nanolaser particle detector. D) Western blot analysis of inclusion and exclusion exosome markers. E) Average cCCT2 copies per exosome as determined by absolute qPCR (n = 6 per group). F) Representative fluorescence microscopy images of DiI‐labeled (red) exosomes taken up by PDAC cells (Capan‐1) and macrophages (THP‐1) (n = 6 per group). The cell nuclei were stained with Hoechst (blue). Scale bar, 50 µm. G) SA‐β‐Gal staining of PDAC cells after SenExo‐cCCT2 intervention and quantification of the results (n = 6 per group). Scale bar, 30 µm. H) Assessment of the effect of SenExo‐cCCT2 intervention on the proliferation of Capan‐1 3D microtumor spheroids derived from cells (n = 6 per group). Scale bar, 100 µm. I) Assessment of the effect of SenExo‐cCCT2 intervention on the apoptosis of patient‐derived organoids (PDO) and quantification of the results (n = 6 per group). Live cells were stained with calcein‐AM (green), and dead cells were stained with PI (red). Scale bar, 50 µm. J) Schematic diagram of the patient‐derived xenograft (PDX) model established on the basis of a humanized immune system model in NSG mice. K) Tumor volume, tumor weight, and survival curves of Hu‐NSG bearing PDX models treated with different combinations of SenExo‐cCCT2, chemotherapy, and anti‐PD‐L1 antibodies (n = 5 per group). L) Representative mIF images of tumors from Hu‐NSG bearing PDX models after treatment with different combinations of SenExo‐cCCT2, FOI, and anti‐PD‐L1 antibodies. Scale bar, 100 µm. M) ELISAs of IFN‐γ and TNF‐α levels in tumors from Hu‐NSG bearing PDX models treated with different combinations of SenExo‐cCCT2, FOI, and anti‐PD‐L1 antibodies (n = 5 per group). One‐way ANOVA with ​Dunnett's post‐hoc test (E–I), one‐way ANOVA with Tukey's post‐hoc test (K, M). Log rank test was used to compare the survival time. Not significant (ns); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. A, J created with BioRender.com.

Article Snippet: The human PDAC cell lines PANC‐1 (RRID: CVCL_0480), BxPC‐3 (RRID: CVCL_0186), Capan‐1 (RRID: CVCL_0237), MIA PaCa‐2 (RRID: CVCL_0428), AsPC‐1 (RRID: CVCL_0152) and SW1990 (RRID: CVCL_1723) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Construct, Transmission Assay, Electron Microscopy, Western Blot, Fluorescence, Microscopy, Labeling, Staining, Derivative Assay

Journal: Frontiers in cellular neuroscience

Article Title: Morphine Binds Creatine Kinase B and Inhibits Its Activity.

doi: 10.3389/fncel.2018.00464

Figure Lengend Snippet: FIGURE 1 | Characterization of the creatine kinase B (CK-B) affinity for morphine. (A) Affinity of mouse recombinant CK-B (rCK-B) for morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) using an immunoenzymatic assay (enzyme-linked immunosorbent assay, ELISA). ELISAs were performed with the 3A6 morphine antibody which detects morphine, codeine, M3G and M6G with the same affinity. Optical density increases with formation of peptide-alkaloid complexes. Data are expressed as Mean ± standard error of the mean (SEM) of triplicates (representative result of n = 3 independent experiments). (B) Characterization of CK-B-morphine complexes resistant to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Left panel, Western Blot analysis showing morphine-immunoreactivity (IR) after incubation of rCK-B with morphine (representative result of n = 3 independent experiments). Right panel, silver staining of a duplicate gel performed in parallel.

Article Snippet: The mouse monoclonal 3A6 anti-morphine antibodies (ref AMM00033; Aviva System Biology) were raised against a ((5 alpha 6 alpha) 7 8 didehydro-4 5 epoxy-17methylmorphinan-3 5diol)-BSA conjugate.

Techniques: Recombinant, Immunoenzymatic Assay, Enzyme-linked Immunosorbent Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Incubation, Silver Staining

Journal: Frontiers in cellular neuroscience

Article Title: Morphine Binds Creatine Kinase B and Inhibits Its Activity.

doi: 10.3389/fncel.2018.00464

Figure Lengend Snippet: FIGURE 2 | Affinity of synthetic peptides generated from mouse CK-B sequence for morphine and its derivatives. (A) Map of the six CK-B-derived peptides. (B) Immunoenzymatic (ELISA) determination of the binding properties of peptides CK-B1–75 and (C) CK-B184–258 to morphine, M6G, M3G and codeine. Data are expressed as Mean ± SEM of triplicates (representative result of n = 3 independent experiments). ELISAs were performed with the 3A6 anti-morphine antibody which binds morphine, codeine, M3G and M6G. Optical density increases with formation of peptide-alkaloid complexes.

Article Snippet: The mouse monoclonal 3A6 anti-morphine antibodies (ref AMM00033; Aviva System Biology) were raised against a ((5 alpha 6 alpha) 7 8 didehydro-4 5 epoxy-17methylmorphinan-3 5diol)-BSA conjugate.

Techniques: Generated, Sequencing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Binding Assay