Journal: bioRxiv

Article Title: Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy

doi: 10.1101/418640

Figure Lengend Snippet: (A)Diagram of the homogeneous time-resolved fluorescence assay. Step 1, the enzymatic reaction: p300 transfers an acetyl group from acetyl-CoA to tau, producing CoA and ac-tau. Step 2, detection: a rabbit ac-tau–specific antibody (mAB359) recognizes ac-tau, the europium cryptate-labeled anti-rabbit IgG-EuK (donor) binds to mAB359, and the anti-GST-D2 (acceptor) captures GST-tau. When tau is acetylated, the Eu donor and D2 acceptor are brought into proximity, allowing Forster resonance energy transfer (FRET) to occur. FRET is detected as a long-lived fluorescence signal at 665 nm. The fluorescence signal ratio 665/620 nm is proportional to the extent of ac-tau in the solution. (B)Work flow of the high-throughput screen. (C)Structure of SMDC37892. (D, E) 37892 inhibits p300 activity in HEK293T. (D) Representative immunoblots of acH3K18 and H3 in histone extracts of HEK293T cells treated with increasing doses of 37892 for 24 h. (E) Concentration-response curve for 37892, quantified from p300 activity in HEK293T cells. (F–J) 37892 treatment in hTau-expressing rat primary neurons. (F) Representative immunoblots of p62, LC3-I, -II and GAPDH in primary neurons treated with 37892 (50 μM) for 24 h. (G) Representative immunoblots of LC3-I, -II and actin in primary neurons treated with 37892 (50 μM), BafA1 (5 nM), or both, for 24 h. (H) Autophagic flux in control and 37892-treated neurons quantified from the increase in LC3-II levels in response to BafA1 treatment, normalized to control. (I) Quantification of intracellular tau levels by ELISA and normalized to control. (J) Quantification of relative tau secretion over 3 h, based on levels of extracellular tau and intracellular tau measured by ELISA and normalized to control. n=6 wells from three independent experiments. **p<0.01 by unpaired t test. Values are mean ± SEM.

Article Snippet: Secondary antibodies include fluorescein-labeled goat anti-mouse IgG and goat anti-rabbit IgG (1:500, Vector Laboratories).

Techniques: Fluorescence, Labeling, Förster Resonance Energy Transfer, High Throughput Screening Assay, Activity Assay, Western Blot, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Prostate Cancer

Article Title: Circulating MicroRNAs as Biomarkers of Prostate Cancer: The State of Play

doi: 10.1155/2013/539680

Figure Lengend Snippet: Studies investigating the potential of circulating miRNAs as biomarkers of prostate cancer.

Article Snippet: Bryant and colleagues assessed the diagnostic capacity of plasma miRNAs using Exiqon's high-throughput qRT-PCR platform [ ].

Techniques: Microarray, Expressing, Diagnostic Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Targeted delivery of platinum-taxane combination therapy in ovarian cancer

doi: 10.1016/j.jconrel.2015.09.007

Figure Lengend Snippet: Tissue distribution of platinum in different treatment groups as determined by ICP-MS. Mice were sacrificed at day 14 of the treatment with FA-[(CDDP+PTX)/NG] IP or FA-[(CDDP+PTX)/NG] or FA+ FA-[(CDDP+PTX)/NG] or (CDDP+PTX)/NG or free CDDP. Data are presented as mean ± SD (n = 3). * P < 0.05.

Article Snippet: Pt content in NGs (Pt194/Pt195) was measured by the inductively coupled plasma-mass spectrometer (ICP-MS, NexION 300Q, ICP-MS spectrometer, PerkinElmer) calibrated with Pt (2–100 ng/ml) and Holmium as the internal standard.

Techniques:

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution overview. ( A ) SNAP25 sub-family proteins and their isoforms (CLUSTAL 2.1 ). ( B ) Co-crystal structure (PDB: 1XTG ) of LC/A (white) and SNAP25 (dark gray). Eight substitutions in LC/A drive SNAP23 specificity (teal) through substrate control loops (pink) alongside prior substitutions (light gray). ( C ) Platform for the directed evolution of SNAP23 substrate specificity. 1. Random or site-directed mutagenesis (e.g., site-saturation); 2. QC by DNA sequencing; 3. High-throughput protein production; 4. Measure V 0 23 and V 0 25 for substrate specificity; 5. Confirmation screens. The most specific and consistent variant from the DARET assay entered the next round of directed evolution. ( D ) Sequence alignment of substrates used for screening (UniProt: P60880, O00161). The SNAP binding exosites in LC/A (residue numbers above) and cleavage site (scissors) are shown. The gradient of color indicates homology from identical (white, *), to strongly similar (light gray, :), weakly similar (teal, .), or dissimilar (dark teal, space).

Article Snippet: After 3 days of differentiation, SiMa-H1 cells were incubated for 24 h with an adenovirus- human type 5 (dE1/E3) vector encoding ORFs for m-Cherry and human SNAP23 under two independent CMV promoters (Vector biolabs) in SFM supplemented with GT1b.

Techniques: Control, Mutagenesis, DNA Sequencing, High Throughput Screening Assay, Variant Assay, Sequencing, Binding Assay, Residue

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution of a SNAP23-specific LC/A. ( A ) Improvement in specificity index through directed evolution in salt-free buffer. The most specific variant from each round was assayed multiple times (replicates shown). The average specificity index (horizontal bars) and standard deviation (error bars) are shown. The final dilution of cell lysates for screening is indicated by color code. ( B ) Increased specificity index achieved through directed evolution in salt buffer. ( C ) Roadmap for directed evolution showing the fold increase in specificity indices over eight rounds (R1 to R8). As shown in Fig. S1, the substrate specificity of the qmLC/A variant is sensitive to screening dilution; therefore, each point represents the variant’s average specificity normalized to the qmLC/A average specificity at the corresponding screening dilution. Advancement to the next round (solid line) weighed specificity index, protein solubility, and stability.

Article Snippet: After 3 days of differentiation, SiMa-H1 cells were incubated for 24 h with an adenovirus- human type 5 (dE1/E3) vector encoding ORFs for m-Cherry and human SNAP23 under two independent CMV promoters (Vector biolabs) in SFM supplemented with GT1b.

Techniques: Variant Assay, Standard Deviation, Solubility

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Enzyme kinetics of LC/A variants.

Article Snippet: After 3 days of differentiation, SiMa-H1 cells were incubated for 24 h with an adenovirus- human type 5 (dE1/E3) vector encoding ORFs for m-Cherry and human SNAP23 under two independent CMV promoters (Vector biolabs) in SFM supplemented with GT1b.

Techniques:

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Characterization of omLC/A cleavage. ( A ) Rates of SNAP cleavage by batch-expressed, purified qmLC/A and ( B ) omLC/A at the indicated DARET substrate concentrations (n = 3). ( C ) Deconvoluted ESI–MS of recombinant, full-length SNAP23 (fl-S23) treated with DTT and iodoacetamide to carbamidomethylate its six cysteines (6 × CAM). The mass spectrum of intact fl-S23 incubated with buffer (top, black) is compared to that of fl-S23 incubated with omLC/A (bottom, teal). Intact fl-S23 (1) and cleaved fl-S23 (cl. fl-S23, 2) peaks are labeled. Additional marked peaks correspond to the masses of peaks 1 and 2 plus one additional CAM (+ 57 Da), likely resulting from overalkylation by iodoacetamide . The cleaved peptide was not directly observed, but inferred from the mass difference of peaks 1 and 2. The deconvolution error is +/- 2 Da. ( D ) Recombinant omBoNT/A was purified by IMAC followed by anion exchange (AEX) chromatography. The omBoNT/A is ≈95% nicked upon DTT reduction as demonstrated by the presence of the HC/A and omLC/A bands. ( E ) In vitro cleavage of recombinant fl-S23 by two independent preparations of omBoNT/A (1 and 2) visualized with a C-terminal anti-SNAP23 antibody; before proteolysis, omBoNT/A was reduced with TCEP. The untreated, wtLC/A, and wtLC/E lanes provide negative controls. ( F ) In cellulo cleavage of SNAP23 and SNAP25 in SiMA-H1 cells infected with adenovirus delivering DNA encoding mCherry/SNAP23. Cells were treated with omBoNT/A or wtBoNT/A or without toxin (ct). Proteins, full-length (fl) or cleaved (cl), were identified by Western blotting with anti-SNAP23, -SNAP25, or -mCherry antibodies (M, MW marker). Full-length images of these gels with multiple exposures where necessary are shown in Fig. S12 and S13).

Article Snippet: After 3 days of differentiation, SiMa-H1 cells were incubated for 24 h with an adenovirus- human type 5 (dE1/E3) vector encoding ORFs for m-Cherry and human SNAP23 under two independent CMV promoters (Vector biolabs) in SFM supplemented with GT1b.

Techniques: Purification, Recombinant, Incubation, Labeling, Chromatography, In Vitro, Infection, Western Blot, Marker

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or g2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003

Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

Techniques: Cloning, Expressing, Biomarker Discovery, Plasmid Preparation, Derivative Assay, Sequencing, Translocation Assay, Membrane

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 2. Cloning of VL and VH domain sequences from hybridomas into the R-mAb expression plasmid. (A) Agarose gel analysis of VL and VH domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG VL and VH domains is » 360 bp. (B) Agarose gel analysis of VH and digested VL fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. (E) Agarose gel analysis of PCR products of VL domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact VL domains are » 360 bp, and the digested aberrant light chains » 180 bp. DOI: https://doi.org/10.7554/eLife.43322.004

Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

Techniques: Cloning, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 3. Validation of subclass-switched anti-PSD-95 K28/43R R-mAb. (A) Validation of the K28/43R R-mAb in heterologous cells. COS-1 cells transiently transfected to express human PSD-95 in a subset of cells were immunolabeled with K28/43 mAb (IgG2a) alone (top row), K28/43R R-mAb (IgG1) alone (middle row), or K28/43 mAb plus K28/43R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG2a (red, for the K28/43 mAb) and anti-mouse IgG1 (green, for the K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A. (B) Validation of the K28/43R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K28/43 mAb plus K28/43R R-mAb and immunolabeling detected with a cocktail of anti-mouse IgG2a (red, for K28/43 mAb) and anti-mouse IgG1 (green, for K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA- specific dye Hoechst 33258 (blue). The region of interest shown is from cerebellar cortex. Scale bar in the left panel = 100 mm, and in the right merged panel = 30 mm. (C) Immunoblots against brain membranes and COS cell lysates over-expressing various members of the MAGUK superfamily of scaffolding proteins. To confirm expression of MAGUK proteins, immunoblots were probed with rabbit anti-PSD-95 (red). K28/86 is an anti-MAGUK mAb. Primary antibodies were detected with the appropriate combinations of fluorescently labeled species-specific anti-rabbit and subclass-specific anti-mouse IgG secondary Abs as indicated. Control indicates COS cells transfected with an empty vector. DOI: https://doi.org/10.7554/eLife.43322.006

Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

Techniques: Biomarker Discovery, Transfection, Immunolabeling, Labeling, Western Blot, Expressing, Scaffolding, Control, Plasmid Preparation

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 4. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. (A) A section from neocortex labeled with anti- pan-Nav R-mAb K58/35R (IgG2a, red) at nodes of Ranvier and AIS (arrows), anti-CASPR mAb K65/35 (IgG1, green) at paranodes, and anti-Kv2.1 rabbit polyclonal (KC) antibody (blue) on somata and proximal dendrites. Scale bar = 150 mm. Insets (dashed box) show details of labeling for pan-Nav (red) and CASPR (green) at the node and paranodes (arrows), respectively, at a single node of Ranvier as indicated by box in main panel. (B) A section Figure 4 continued on next page

Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

Techniques: Multiplex Assay, Immunolabeling, Recombinant, Labeling

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 5. Cloning of anti-Kv2.1 D3/71 VL and VH domain cDNAs from a nonviable hybridoma. (A) Agarose gel analysis of PCR amplified VL and VH domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the VL after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG VL and VH domains is » 360 bp, and of the cleaved aberrant VL domain is » 180 bp. (B) Agarose gel analysis of D3/71 VH and digested VL fragments joined by fusion PCR (F- PCR) to the P1316 joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. DOI: https://doi.org/10.7554/eLife.43322.008

Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

Techniques: Cloning, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay, Plasmid Preparation

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 6. Recovery of a functional anti-Kv2.1 D3/71R R-mAb from nonviable hybridomas. (A) Validation of the D3/71R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), D3/71R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus D3/71R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. (B) Validation of the subclass-switched K89/34R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), K89/34R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus K89/34R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the K89/34R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue in panels A and B is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A and B. (C) Validation of the D3/71R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K89/34 mAb plus D3/71 R-mAb and the immunolabeling detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). Region of interest shown is from neocortex. Scale bar = 30 mm. (D) Strip immunoblots on a crude rat brain membrane fraction immunolabeled with the K89/34 mAb, the K89/34R R-mAb, and the D3/71 R-mAb as indicated. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and ECL. DOI: https://doi.org/10.7554/eLife.43322.009

Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

Techniques: Functional Assay, Biomarker Discovery, Transfection, Immunolabeling, Labeling, Stripping Membranes, Western Blot, Membrane, Autoradiography

Journal: bioRxiv

Article Title: Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy

doi: 10.1101/418640

Figure Lengend Snippet: (A)Diagram of the homogeneous time-resolved fluorescence assay. Step 1, the enzymatic reaction: p300 transfers an acetyl group from acetyl-CoA to tau, producing CoA and ac-tau. Step 2, detection: a rabbit ac-tau–specific antibody (mAB359) recognizes ac-tau, the europium cryptate-labeled anti-rabbit IgG-EuK (donor) binds to mAB359, and the anti-GST-D2 (acceptor) captures GST-tau. When tau is acetylated, the Eu donor and D2 acceptor are brought into proximity, allowing Forster resonance energy transfer (FRET) to occur. FRET is detected as a long-lived fluorescence signal at 665 nm. The fluorescence signal ratio 665/620 nm is proportional to the extent of ac-tau in the solution. (B)Work flow of the high-throughput screen. (C)Structure of SMDC37892. (D, E) 37892 inhibits p300 activity in HEK293T. (D) Representative immunoblots of acH3K18 and H3 in histone extracts of HEK293T cells treated with increasing doses of 37892 for 24 h. (E) Concentration-response curve for 37892, quantified from p300 activity in HEK293T cells. (F–J) 37892 treatment in hTau-expressing rat primary neurons. (F) Representative immunoblots of p62, LC3-I, -II and GAPDH in primary neurons treated with 37892 (50 μM) for 24 h. (G) Representative immunoblots of LC3-I, -II and actin in primary neurons treated with 37892 (50 μM), BafA1 (5 nM), or both, for 24 h. (H) Autophagic flux in control and 37892-treated neurons quantified from the increase in LC3-II levels in response to BafA1 treatment, normalized to control. (I) Quantification of intracellular tau levels by ELISA and normalized to control. (J) Quantification of relative tau secretion over 3 h, based on levels of extracellular tau and intracellular tau measured by ELISA and normalized to control. n=6 wells from three independent experiments. **p<0.01 by unpaired t test. Values are mean ± SEM.

Article Snippet: After overnight incubation, the sections were incubated with secondary antibodies including Cy3-labeled donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch) and fluorescein-labeled goat anti-mouse IgG (1:500, Vector Laboratories).

Techniques: Fluorescence, Labeling, Förster Resonance Energy Transfer, High Throughput Screening Assay, Activity Assay, Western Blot, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature communications

Article Title: Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

doi: 10.1038/s41467-019-08810-0

Figure Lengend Snippet: Fig. 2 High-throughput, siRNA-based screening to identify cellular factors regulating HIV-1 integrase stability. a Workflow for the siRNA-based screening. Cellular fluorescence, as surrogate of IN levels, was analyzed by automated, high-content fluorescent microscopy. b Results of screening. The graphs show the log10 values of the fold change of EGFP-positive cells over control in the two replicate screenings (R1 and R2). The dotted lines show 2x increase over Control (pool of results using 4 non-targeting siRNAs and mock-transfected cells). The 6 siRNAs in red are those that were in the top 10 in both screenings. The 4 siRNAs in green are those that were among the top 10 in one of screening while anyhow showing an effect ≥2 fold over control in the other screening. The effect of MG132 is shown in blue. c Confirmation of effective silencing of Pin1, TRIM33, FBOX28, RNF31, RNF125, RFPl3, and DTX by immunoblotting with the respective antibodies. Cells transfection of non-targeting siRNA1 (siNT1) was used as a control. HSC70 served as a loading control. d Representative immunoblot showing the levels of HIV-IN after knock-down of the top 10 E3 ligases from the screening. HeLa cells were transfected with siRNAs against the identified factors or with a siRNA against Pin1, followed by transfection of Flag-IN and EGFP. Forty-eight hour later the levels of IN were assessed by anti-Flag immunoblotting. Tubulin served as a loading control; β-catenin was used to confirm effect of the MG132 treatment. The bottom panel shows a western blotting for EGFP in a representative experiment to show lack of effect of any of the tested siRNAs on EGFP levels. e Quantification of the levels of HIV-1 IN after knock-down of the top 10 E3 ligases identified by the screening. Experiments were performed as in panel d. IN levels are expressed after normalization for tubulin and as fold over siNT1. Data are mean ± SEM; n = 3 independent experiments; *P < 0.05; **P < 0.01; one-way ANOVA. f Representative high-content microscopy images showing of EGFP-IN-expressing cells after depletion of four cellular ubiquitin- conjugation factors (TRIM33, FBXO28, siDTX2, and siUBE2J2) or Pin1

Article Snippet: The lentiviral vector expressing the TRIM33 cDNA was kindly provided by David A. Calderwood (Yale University School of Medicine, New Haven, USA); other TRIM33 lentiviral vectors were purchased from Open Biosystem (Huntsville, AL) and control pLKO.1based scramble lentiviral vector was purchased from Addgene.

Techniques: High Throughput Screening Assay, Microscopy, Control, Transfection, Western Blot, Knockdown, Expressing, Ubiquitin Proteomics, Conjugation Assay

Journal: Nature communications

Article Title: Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

doi: 10.1038/s41467-019-08810-0

Figure Lengend Snippet: Fig. 3 Cellular TRIM33 is only TIF1 family member that negatively regulates HIV-1 integrase stability. a Schematic representation of domain organization of TIF1 subfamily members. b Effect of anti-TRIM24 siRNAs. The immunoblots show the levels of TRIM24, TRIM33, TRIM28, and HIV-1 IN in HeLa cells transfected with individual siRNAs against TRIM24 (#4, #5, #6, and #7), their pool (Pool) or non-targeting siRNA-2 (siNT2). MG132 was used to assess the effect of inhibiting proteasome-mediated protein degradation. c Effect of anti-TRIM28 siRNAs. Same as in panel b using siRNAs against TRIM28. d Effect of anti-TRIM33 siRNAs. Same as in panel b using siRNAs against TRIM33. e Stability of HIV-1 IN protein in siRNA-treated cells. Representative immunoblots showing the levels of HIV-1 IN after HeLa cell transfection with siRNAs against TRIM33, Pin1, and LEGF/p75 at different times and treatment with cycloheximide (30 µg/ml) to block protein degradation. Tubulin served as a loading control. f Quantification of HIV-1 IN levels. Experiments were performed as in panel e. IN amounts are normalized to Tubulin and expressed as percent over IN at time = 0 for each treatment. Data are mean ± SEM; n = 3 independent experiments. g Effects of siRNAs on the levels of HIV-1 and MLV IN proteins. The figure shows representative immunoblots using lysates of HeLa cells transfected to express either Flag-IN from HIV-1 (upper part) or MLV-IN (lower part)and siRNAs against the indicated factors. Tubulin and HSC70 served as loading controls. h Quantification of the effects of siRNAs against the indicated factors or treatment with MG132 on the levels of HIV-1 IN. IN levels are expressed after normalization for tubulin and as fold over non-targeting siRNA siNT1. Data are mean ± SEM; n = 3 independent experiments; *P < 0.05; **P < 0.01; one-way ANOVA. i Same as in panel g for MLV-IN. Normalization was for cellular HSC70. Data are mean ± SEM; n = 3 independent experiments; *P < 0.05; one-way ANOVA

Article Snippet: The lentiviral vector expressing the TRIM33 cDNA was kindly provided by David A. Calderwood (Yale University School of Medicine, New Haven, USA); other TRIM33 lentiviral vectors were purchased from Open Biosystem (Huntsville, AL) and control pLKO.1based scramble lentiviral vector was purchased from Addgene.

Techniques: Western Blot, Transfection, Blocking Assay, Control

Journal: Nature communications

Article Title: Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

doi: 10.1038/s41467-019-08810-0

Figure Lengend Snippet: Fig. 4 Cellular TRIM33 binds HIV-1 integrase and regulates its degradation by poly-ubiquitination. a Endogenous TRIM33 interacts with HIV-1 IN in vivo. HEK293T cells were transfected with the plasmids indicated on the top of the panel and immunoprecipitated with anti-Flag M2 beads, followed by immunoblotting with anti-TRIM33 antibody (upper blot). The lower three blots show the levels of expression of the transfected proteins in cell lysates. b Schematic representation of the HIV-1 IN domains used for the pull-down assay in panel c. NTD N-terminal domain, CCD catalytic core domain, CTD C-terminal domain. c TRIM33 binds the HIV-1 IN C-terminal domain (CTD) in in vitro pull-down assay. The indicated fragments of the HIV-1 IN protein fused to GST or GST alone were incubated with in vitro translated [35S]-TRIM33, extensively washed, and then resolved by SDS-PAGE. The middle pictures shows the gel exposed to a phosphoimager from a representative experiment along with the quantification of the amount of bound proteins expressed as a percentage of radiolabeled input. The lower panel shows the protein gel stained with Coomassie blue to visualize proteins. Quantification in the upper graph reports mean ± SEM; n = 3 independent experiments; **P < 0.01; one-way ANOVA. d HIV-1 IN is degraded through Lys48-linked polyubiquitination. HEK293T cells were transfected with Flag-IN and HA-Ubiquitin for 48 h, as indicated. The ubiquitination profile of IN was visualized after immunoprecipitation with anti-Flag M2 beads using anti-ubiquitin, anti-K48, anti-K63, anti-HA, and anti-Flag antibodies. e TRIM33 Inhibition reduces IN polyubiquitination. HEK293T cells were co-transfected with Flag-IN and HA-Ubiquitin for 36 h, as indicated. IN polyubiquitination was visualized after immunoprecipitation with anti-Flag M2 beads using an anti-IN antibody. f Quantifications of the amount of polyubiquitinated IN protein (Ubi-IN) in whole cell extracts, normalized over total IN. Experiments were performed as in panel e

Article Snippet: The lentiviral vector expressing the TRIM33 cDNA was kindly provided by David A. Calderwood (Yale University School of Medicine, New Haven, USA); other TRIM33 lentiviral vectors were purchased from Open Biosystem (Huntsville, AL) and control pLKO.1based scramble lentiviral vector was purchased from Addgene.

Techniques: Ubiquitin Proteomics, In Vivo, Transfection, Immunoprecipitation, Western Blot, Expressing, Pull Down Assay, In Vitro, Incubation, SDS Page, Staining, Inhibition

Journal: Nature communications

Article Title: Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

doi: 10.1038/s41467-019-08810-0

Figure Lengend Snippet: Fig. 5 Mapping the TRIM determinants determining integrase stability. a Schematic representation of the TRIM33 protein and its catalytically inactive mutants in the RING and PHD domains [RING(CA) and PHD(CA) respectively]. b Integrity of the TRIM33 RING motif is essential for HIV-1 IN ubiquitination. HEK293T cells were transfected with combinations of plasmids expressing HA-ubiquitin, EGFP-IN, Flag-TRIM33 and its catalytically inactive mutants (RING and PHD), and pcDNA3 as an empty vector. Cells were harvested after 5 h treatment with proteasome inhibitor and cell lysates were immunoprecipitated with anti-GFP antibody. Ubiquitin-conjugated IN (Ubi-IN) was detected by immunoblotting with anti-HA antibody (upper picture). The lower pictures show the levels of expression of the transfected plasmids in whole cell lysates control expression of transfected proteins after immunoblotting with the respective antibodies. c Quantifications of the amount of ubiquitinated IN protein (Ubi-IN) in whole cell extracts, normalized over total IN. Experiments were performed as in panel b. Data are mean ± SEM; n = 3 independent experiments; *P < 0.05; **P < 0.01; one-way ANOVA. d Representative immunoblot showing the stability of HIV-IN in HeLa cells stably expressing an shRNA against TRIM33 (TRIM33 KD) and transfected to overexpress wild-type TRIM33 or the C125A/C128A catalytically inactive (RING(CA). Cells were co-transfected with Flag-IN and EGFP, as transfection and specificity control; 48 h later, the levels of IN were assessed by anti-Flag immunoblotting. Tubulin served as a loading control; β-catenin was used to confirm effect of MG132 treatment; knock-down and overexpression of TRIM33 variants were verified by anti-TRIM33 immunoblotting. e Quantification of the effects of TRIM33 knock down and overexpression on the levels of HIV-1 IN. Results of three independent experiments (mean ± SEM) performed as in panel d are expressed, after normalization for Tubulin, as a percentage of time = 0 for each treatment

Article Snippet: The lentiviral vector expressing the TRIM33 cDNA was kindly provided by David A. Calderwood (Yale University School of Medicine, New Haven, USA); other TRIM33 lentiviral vectors were purchased from Open Biosystem (Huntsville, AL) and control pLKO.1based scramble lentiviral vector was purchased from Addgene.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Stable Transfection, shRNA, Knockdown, Over Expression

Journal: Nature communications

Article Title: Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

doi: 10.1038/s41467-019-08810-0

Figure Lengend Snippet: Fig. 6 TRIM33 inhibits HIV-1 infection. a Analysis of the effect of stable knockdown of TRIM33 in multiple round infection with wild-type HIV-1. SupT1 cells were first transduced with lentiviral vector expressing different anti-TRIM33 shRNAs; after selection with puromycin, cells were infected with wild-type HIV-1BRU or HIV-1BRUIN(S57A), followed by analysis of infection over time by p24 ELISA. b Western blotting analysis showing endogenous TRIM33 levels in SupT1 cells transduced with lentiviral vectors expressing the indicated shRNAs and selected with puromycin. Tubulin was used as a loading control. c. Stable knockdown of TRIM33 enhances wild type HIV-1 replication and rescues replication of the IN(S57A) mutant. The graph show the levels of p24 in control Sup1 cells or cells previously transduced with anti-TRIM33 shRNA #4, which efficiently knocks down TRIM33 expression, after infection with the two viruses. The results shown are representative or four independent experiments. d Quantification of the levels of integrated HIV-1 DNA in SupT1 cells, expressing an shRNA control or shRNA #4 against TRIM33, and infected with wild-type HIV-1BRU or HIV-1BRUIN(S57A), at day 3 after infection. Data are mean ± SEM; n = 3 independent experiments; **P < 0.01; n.s. not significant; one-way ANOVA. e Silencing of TRIM33 protects both wild-type IN and mutant IN(S57A) from degradation. Expression plasmids for the two IN proteins were transfected into HeLa cells in which TRIM33 was knocked down by RNAi; cells were then treated with cycloheximide 30 µg/ml for the indicated time points prior to lysis. The panel shows a representative immunoblots using antibodies against IN, TRIM33 and tubulin as a loading control. f Quantification of IN levels in the presence of cycloheximide for the indicated time points in the whole cell extracts of SupT1 cells treated with siRNAs against TRIM33 or not targeting and transfected with either wt IN or IN(S57A). After normalization for Tubulin, data (mean ± SEM; n = 3 independent experiments) are expressed as a percentage of time = 0 for each treatment

Article Snippet: The lentiviral vector expressing the TRIM33 cDNA was kindly provided by David A. Calderwood (Yale University School of Medicine, New Haven, USA); other TRIM33 lentiviral vectors were purchased from Open Biosystem (Huntsville, AL) and control pLKO.1based scramble lentiviral vector was purchased from Addgene.

Techniques: Infection, Knockdown, Transduction, Plasmid Preparation, Expressing, Selection, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Mutagenesis, shRNA, Transfection, Lysis

Journal: Nature communications

Article Title: Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

doi: 10.1038/s41467-019-08810-0

Figure Lengend Snippet: Fig. 7 Interaction of TRIM33 with integrase from infectious HIV-1. a Analysis of IN protein levels in TRIM33-knockdown (TRIM33 KD) cells during HIV-1 infection. Cells stably expressing an anti-TRIM33 shRNA were infected for 1 h with HIV-1; at the indicated time points after infection, cells were washed with PBS and the levels of IN were analyzed. b Western blot showing IN levels in control and TRIM33 depleted cells Beta-actin was used as a loading control; TRIM33 downregulation was verified using an anti-TRIM33 antibody. c TRIM33 interacts with IN during HIV-1 infection. SupT1 cells were infected with HIV-1 in the presence of MG132. Either TRIM33 or IN were immunoprecipitated with the respective antibodies, followed by immunoblotting to test the presence of the reciprocal protein. d Analysis of the effect of stable knockdown and overexpression of TRIM33 variants in multiple round infection with wild-type HIV-1. SupT1 cells stably expressing an hRNA targeting the TRIM33 3′-UTR (selected after transduction with a lentiviral vector and selection for puromycin) were transduced with lentiviral vectors expressing the TRIM33 coding sequence (cs) or the coding sequence of the RING(CA) mutant. After selection with hygromycin, cells were infected with wild type HIV-1BRU, followed by analysis of infection over time by p24 ELISA. e Western blotting analysis showing TRIM33 levels after transduction with shRNA targeting 3′-UTR of TRIM33 alone or after transduction of wild-type or mutant TRIM33 (TRIMM33cs or RING(CA)cs. Tubulin was used as a loading control. f real-Time RT-PCR analysis showing TRIM33 expression after transduction with the shRNA targeting the 3′-UTR of TRIM33 alone or after transduction of wild type or mutant TRIM33. g Stable knockdown of TRIM33 enhances wild-type HIV-1 replication, while overexpression of its catalytically active cDNA reduces it. The graph shows the levels of p24 in control Sup1 cells or cells previously transduced with anti-TRIM33 shRNA targeting the 3′-UTR of the protein, with or without transduction with a lentiviral vector expressing the coding sequence of wild type TRIM33 (TRIM33cs) or that of the catalytically inactive RING(CA) mutant (RING(CA)cs), after infection with wild-type virus. The results are representative of three independent experiments

Article Snippet: The lentiviral vector expressing the TRIM33 cDNA was kindly provided by David A. Calderwood (Yale University School of Medicine, New Haven, USA); other TRIM33 lentiviral vectors were purchased from Open Biosystem (Huntsville, AL) and control pLKO.1based scramble lentiviral vector was purchased from Addgene.

Techniques: Knockdown, Infection, Stable Transfection, Expressing, shRNA, Western Blot, Control, Immunoprecipitation, Over Expression, Transduction, Plasmid Preparation, Selection, Sequencing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Virus

Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a Schematic of the murine Il33 genomic locus depicting the two transcriptional isoforms with a highlighted 800 bp region of the Il33-002 promoter and the locations of the 11 CpGs as well as four gRNAs targeting specific CpGs. The 11 CpGs are numbered sequentially in the 5′ to 3′ direction. The promoter-targeting gRNAs used in these experiments are shown relative to the CpGs and are approximately to scale such that CpGs 1, 2, and 3 are targeted by gRNA1, CpG 5 by gRNA 2, and gRNA 3 targets CpGs 9, 10, and 11—which overlap the transcription start site (TSS), marked by a black arrow. The orientation of the gRNAs is indicated by the direction of the arrow labeled with the respective gRNA, where an arrow pointing to the left indicates a gRNA that binds the plus strand. The fragment cloned into the luciferase vector (pCpGl) is marked at either end, spanning from −844 to +171 relative to the TSS. b Percent of DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing at the three TSS CpGs (labeled 9–11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 independent experiments for CpGs 1, 2, 3, and 5; n = 6 for CpGs 9, 10, and 11). c Expression of Il33-002 (mean ± SEM) quantified by RT-qPCR and normalized to beta actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 biologically independent samples) (Student’s t -test, two sided, control vs. 0.1 µM 5-aza; P = 0.1636, control vs. 1 µM 5-aza; P = 0.0482). d Expression (mean ± SEM) of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 biologically independent samples). e – g Percent of DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing at seven targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 ( e ), gRNA2 ( f ), or gRNA3 ( g ) compared to gRNAscr (light and dark gray, gRNAscr data identical in e – g and shown for comparison) ( n = 4–8 biologically independent experiments, depending on specific condition and CpG; see Source Data file for specific n of interest). h , i Expression of Il33-002 ( h ) and Il33-001 ( i ) (mean ± SEM) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET ( n = 3–4 biologically independent samples; statistical comparisons are between each gRNA and gRNAscr bearing the same dCas9 construct (dCas9-TET or dCas9-deadTET)). All data shown as (mean± SEM). j Relative light units normalized to protein quantity (mean ± SEM) in transfected HEK293 cells. Cells were transiently transfected with methylated or unmethylated SV40-luciferase vector along with mammalian wild-type or mutant human TET1 expression plasmid or empty vector (pEF1A) control ( n = 3). * indicates statistically significant difference of P < 0.05, ** P < 0.01, *** P < 0.001, **** or # of P < 0.0001, and ns not significant (Student’s t -test, two-sided, with Holm-Sidak correction if number of tests is greater than 3). Source data are provided as a Source Data file.

Article Snippet: The fragment cloned into the luciferase vector (pCpGl) is marked at either end, spanning from −844 to +171 relative to the TSS. b Percent of DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing at the three TSS CpGs (labeled 9–11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 independent experiments for CpGs 1, 2, 3, and 5; n = 6 for CpGs 9, 10, and 11). c Expression of Il33-002 (mean ± SEM) quantified by RT-qPCR and normalized to beta actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 biologically independent samples) (Student’s t -test, two sided, control vs. 0.1 μM 5-aza; P = 0.1636, control vs. 1 μM 5-aza; P = 0.0482). d Expression (mean ± SEM) of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 biologically independent samples). e – g Percent of DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing at seven targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 ( e ), gRNA2 ( f ), or gRNA3 ( g ) compared to gRNAscr (light and dark gray, gRNAscr data identical in e – g and shown for comparison) ( n = 4–8 biologically independent experiments, depending on specific condition and CpG; see Source Data file for specific n of interest). h , i Expression of Il33-002 ( h ) and Il33-001 ( i ) (mean ± SEM) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET ( n = 3–4 biologically independent samples; statistical comparisons are between each gRNA and gRNAscr bearing the same dCas9 construct (dCas9-TET or dCas9-deadTET)).

Techniques: Labeling, Clone Assay, Luciferase, Plasmid Preparation, DNA Methylation Assay, Control, Expressing, Quantitative RT-PCR, Transduction, Selection, Infection, Flow Cytometry, Construct, Comparison, Stable Transfection, Transfection, Methylation, Mutagenesis

Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a Pyrosequencing data (mean ± SEM, n = 4 biologically independent samples) for the methylation state of indicated CpGs in the Il33 -pCpGl plasmid following standard methylation for 4 h by M.SssI (dark gray), methylation in the presence of dCas9 and gRNA 6 (distant binding) (gray), or a mock-methylated control reaction that lacked S-adenosyl methionine substrate (light gray). b Diagram illustrating the principle of site-specific methylation utilizing pre-incubation of DNA with dCas9 and selective CpG-targeting guide restricting M.SssI from binding and methylating the targeted region, while permitting methylation of remaining unobstructed CpGs. c – e Pyrosequencing data ( n = 4 biologically independent samples, mean ± SEM)) for the methylation state of CpGs in the IL-33-pCpGl plasmid following pre-incubation with dCas9 and gRNA1 ( c ), gRNA2 ( d ), or gRNA3 ( e ) and methylation by M.SssI (colored bars). Gray bars are identical ( a , c – e ) and indicate methylation levels for the same treatment utilizing gRNA6. f Luciferase reporter activity of the plasmids ( a , c – e ), expressed as relative light units (mean ± SEM) normalized for protein content per sample, and then normalized to average value for mock methylated condition ( n = 3 biologically independent experiments). All statistical comparisons are to mock methylated conditions unless otherwise indicated. g Percent of methylation (mean ± SEM) assayed by pyrosequencing when Il33-pCpGl is incubated with dCas9 and gRNA 3 or only gRNA 3 (no dCas9 control) after standard methylation, instead of before ( n = 3 biologically independent samples). * indicates statistically significant difference of P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns not significant (Student’s t -test, two-sided, with Holm-Sidak correction if number of tests is greater than 3). Source data are provided as a Source Data file.

Article Snippet: The fragment cloned into the luciferase vector (pCpGl) is marked at either end, spanning from −844 to +171 relative to the TSS. b Percent of DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing at the three TSS CpGs (labeled 9–11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 independent experiments for CpGs 1, 2, 3, and 5; n = 6 for CpGs 9, 10, and 11). c Expression of Il33-002 (mean ± SEM) quantified by RT-qPCR and normalized to beta actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 biologically independent samples) (Student’s t -test, two sided, control vs. 0.1 μM 5-aza; P = 0.1636, control vs. 1 μM 5-aza; P = 0.0482). d Expression (mean ± SEM) of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2′-deoxycytidine (5-aza) or water control ( n = 3 biologically independent samples). e – g Percent of DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing at seven targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 ( e ), gRNA2 ( f ), or gRNA3 ( g ) compared to gRNAscr (light and dark gray, gRNAscr data identical in e – g and shown for comparison) ( n = 4–8 biologically independent experiments, depending on specific condition and CpG; see Source Data file for specific n of interest). h , i Expression of Il33-002 ( h ) and Il33-001 ( i ) (mean ± SEM) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET ( n = 3–4 biologically independent samples; statistical comparisons are between each gRNA and gRNAscr bearing the same dCas9 construct (dCas9-TET or dCas9-deadTET)).

Techniques: Methylation, Plasmid Preparation, Binding Assay, Control, Incubation, Luciferase, Activity Assay