Journal: Molecular cancer research : MCR

Article Title: USP22 interacts with PALB2 and promotes chemotherapy resistance via homologous recombination of DNA double strand breaks

doi: 10.1158/1541-7786.MCR-19-0053

Figure Lengend Snippet: Both USP22 and H2BK120ub are necessary for efficient recruitment of key HR factors to DSBs. a. U2OSFOKI cells were transfected with GFP-USP22 and treated with 4OHT and SHIELD1 ligand to induce DDR at 256X LacO array by the endonuclease mCherry-LacI-FOKIWT. Catalytically dead D450A version was used as a negative control. Percentage of cells with GFP-USP22 recruitment to array are indicated with +/− SD. White scale bars indicate 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. b. U2OSFOKI cells treated with 4OHT and SHIELD1 ligand to induce DDR at the 256X LacO array by the endonuclease mCherry-LacI-FOKIWT with/without USP22 siRNA knockdown for 48 hours. Cells were stained with antibodies against indicated proteins. Percentage of cells with recruitment of indicated protein to the array are indicated with +/−SD. White scale bars indicate 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate c. Western blot against endogenous USP22 and β-Actin (loading control) after 48-hour siRNA knockdown, refers to cells from 2b. Numbers to left of western blots indicate molecular weight. d. U2OSFOKI cells were transfected with GFP-USP22 and indicated FLAG-H2B type and treated with 4OHT and SHIELD1 ligand to induce DDR at 256X LacO array by the endonuclease mCherry-LacI-FOKIWT. Percentage of cells with GFP-USP22 recruitment to array are indicated with +/− SD. White scale bars indicate 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. e. U2OSFOKI cells were transfected with indicated FLAG-H2B type for 48 hours then treated with 4OHT and SHIELD1 ligand to induce DDR at the 256X LacO array by the endonuclease mCherry-LacI-FOKIWT. Cells were stained with antibodies against indicated proteins. Percentage of cells with recruitment of indicated protein to the array are indicated with +/−SD. White scale bars indicate 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. For all statistical analysis a student’s two-tailed t-test was used to establish p-value.

Article Snippet: Antibodies: PALB2 (Abcam, ab220861), BRCA2 (Abcam, ab27976), Rad51 (Cell Signaling Technology, 8875S), USP22 (Santa Cruz, sc-390585), FLAG (Sigma, F3165-1MG), β-Actin (Cell Signaling Technology, 4970S), mCherry (Origene, TA180028), yH2A.X (Cell Signaling Technology, 2577S), MBP (New England Biolabs, E8032S), 6X-His (Santa Cruz, sc-8036), ENY2 (Abcam, ab183622), ATXN7L3 (Bethyl, A302-800A), ATXN7 (Bethyl, A302-638A).

Techniques: Transfection, Negative Control, Staining, Western Blot, Molecular Weight, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: USP22 interacts with PALB2 and promotes chemotherapy resistance via homologous recombination of DNA double strand breaks

doi: 10.1158/1541-7786.MCR-19-0053

Figure Lengend Snippet: USP22 modulates PALB2 protein stability aiding in chemoresistance. a. Western blot of H1299 cells against endogenous USP22, BRCA2, PALB2, Rad51, and β-Actin (loading control) after 48-hour siRNA knockdown. Numbers to left of western blots indicate molecular weight. b. U2OSFOKI cells treated with 4OHT and SHIELD1 ligand to induce DDR at the 256X LacO array by the endonuclease mCherry-LacI-FOKIWT with/without USP22 siRNA knockdown for 48 hours and/or transfection of empty vector or FLAG-USP22 plasmids. Cells were stained with antibodies against indicated proteins. Percentage of cells with recruitment of indicated protein to the array are indicated with +/−SD. White scale bars indicate 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. c. Western blot against endogenous USP22 and β-Actin (loading control) after 48-hour siRNA knockdown, refers to cells from 5b. Numbers to left of western blots indicate molecular weight. (Right) western blot to deduce expression of indicated FLAG-USP22 construct. Western blot done against FLAG and β-Actin (loading control). Numbers to left of western blots indicate molecular weight. d. H1299 or H1299FLAG-PALB2 cells stably overexpressing indicated protein had siRNA KD performed then 3uM/6uM cisplatin or DMSO was added in for an additional 72 hours. Cells were washed then counted. Graph indicates total % of living cells as they were pertaining to the control for both cell lines. Experiment was done in triplicate. e. Western blot for cells from Figure 5d against endogenous USP22, PALB2, and β-Actin (loading control). FLAG antibody was used to assay for FLAG-PALB2. Numbers to left of western blots indicate molecular weight.

Article Snippet: Antibodies: PALB2 (Abcam, ab220861), BRCA2 (Abcam, ab27976), Rad51 (Cell Signaling Technology, 8875S), USP22 (Santa Cruz, sc-390585), FLAG (Sigma, F3165-1MG), β-Actin (Cell Signaling Technology, 4970S), mCherry (Origene, TA180028), yH2A.X (Cell Signaling Technology, 2577S), MBP (New England Biolabs, E8032S), 6X-His (Santa Cruz, sc-8036), ENY2 (Abcam, ab183622), ATXN7L3 (Bethyl, A302-800A), ATXN7 (Bethyl, A302-638A).

Techniques: Western Blot, Molecular Weight, Transfection, Plasmid Preparation, Staining, Expressing, Construct, Stable Transfection

Journal: Molecular cancer research : MCR

Article Title: USP22 interacts with PALB2 and promotes chemotherapy resistance via homologous recombination of DNA double strand breaks

doi: 10.1158/1541-7786.MCR-19-0053

Figure Lengend Snippet: USP22 DUB domain interacts with PALB2 WD40 domain. a. Cartoon of different domains of USP22 and PALB2 (upper). Computer model using ZDOCK predicting USP22 DUB (blue) domain binding to PALB2 WD40 domain (red). b. Immunoprecipitation of FLAG-PALB2 plasmid transiently transfected into H1299 cells. Western blot against FLAG (FLAG-PALB2), USP22, ENY2, ATXN7L3, ATXN7, and β-Actin as a loading control in the input. Numbers to the left of western blots indicate molecular weight. c. U2OSFOKI cells co-transfected with the indicated mCherry-LacI protein to tether it to 256X LacO array along with GFP-USP22. mCherry-LacI alone is used as a negative control. Percentage of cells with recruitment of GFP-USP22 to LacO array are indicated with +/− SD. White scale bars indicated 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. d. U2OSFOKI cells co-transfected with mCherry-LacI-PALB2WD40 protein to tether it to 256X LacO array along with indicated GFP-USP22 fragment. Percentage of cells with recruitment of GFP-USP22 to LacO array are indicated with +/− SD. White scale bars indicated 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. For all statistical analysis a student’s two-tailed t-test was used to establish p-value.

Article Snippet: Antibodies: PALB2 (Abcam, ab220861), BRCA2 (Abcam, ab27976), Rad51 (Cell Signaling Technology, 8875S), USP22 (Santa Cruz, sc-390585), FLAG (Sigma, F3165-1MG), β-Actin (Cell Signaling Technology, 4970S), mCherry (Origene, TA180028), yH2A.X (Cell Signaling Technology, 2577S), MBP (New England Biolabs, E8032S), 6X-His (Santa Cruz, sc-8036), ENY2 (Abcam, ab183622), ATXN7L3 (Bethyl, A302-800A), ATXN7 (Bethyl, A302-638A).

Techniques: Binding Assay, Immunoprecipitation, Plasmid Preparation, Transfection, Western Blot, Molecular Weight, Negative Control, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: USP22 interacts with PALB2 and promotes chemotherapy resistance via homologous recombination of DNA double strand breaks

doi: 10.1158/1541-7786.MCR-19-0053

Figure Lengend Snippet: PALB2WD40 directly binds USP22 and stimulates its catalytic DUB activity. a. In-silico model using ZDOCK of USP22DUB binding PALB2WD40 predicting key residues involved in the interaction. b. U2OSFOKI cells co-transfected with the indicated mCherry-LacI protein to tether it to 256X LacO array along with GFP-USP22. Percentage of cells with recruitment of GFP-USP22 to LacO array are indicated with +/− SD. White scale bars indicated 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. c. Immunoprecipitation of mcherry-PALB2 fragments plasmids transiently transfected into H1299 cells. Western blot against mCherry, FLAG (FLAG-USP22), Rad51. mCherry alone is used as a negative control. Numbers to the left of western blots indicate molecular weight. d. In-vitro pulldown using recombinant MBP-PALB2WD40 (prey) and His-USP22FL (bait). MBP was also incubated with His-USP22 as a negative control. Western blot was performed against His (His-USP22) and MBP (MBP-PALB2WD40, MBP). Numbers to the left of western blots indicate molecular weight. e. Ubiquitin cleavage assay using rhodamine-ubiquitin that is quenched and upon cleavage of ubiquitin fluoresces. Proteins indicated below were pre-incubated to form their respective complexes then rhodamine-ubiquitin was added. Units on vertical axis indicate raw fluorescent units at 565nm. Buffer alone was used as a negative control for auto-fluorescence baseline. Error bars indicated +/− SD. Experiment was done in triplicate.

Article Snippet: Antibodies: PALB2 (Abcam, ab220861), BRCA2 (Abcam, ab27976), Rad51 (Cell Signaling Technology, 8875S), USP22 (Santa Cruz, sc-390585), FLAG (Sigma, F3165-1MG), β-Actin (Cell Signaling Technology, 4970S), mCherry (Origene, TA180028), yH2A.X (Cell Signaling Technology, 2577S), MBP (New England Biolabs, E8032S), 6X-His (Santa Cruz, sc-8036), ENY2 (Abcam, ab183622), ATXN7L3 (Bethyl, A302-800A), ATXN7 (Bethyl, A302-638A).

Techniques: Activity Assay, In Silico, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Negative Control, Molecular Weight, In Vitro, Recombinant, Incubation, Cleavage Assay, Fluorescence

Journal: Ecotoxicology and environmental safety

Article Title: Additive cardiotoxicity of a bisphenol mixture in zebrafish embryos: The involvement of calcium channel and pump.

doi: 10.1016/j.ecoenv.2023.115225

Figure Lengend Snippet: Fig. 4. Effects of BPs on cardiomyocyte development. Representative confocal microscopy image of TG(myl7:NLS-mCherry) ZFEs at 48 hpf exposed to 0.05% DMSO (A) or Mix 2 (BPA 8.7 µM + BPF 18 µM + BPAF 1.3 µM + BPB 4.2 µM), which induced approximately 40% heart rate reduction (B). BP exposure did not affect the cardiomyocyte count (C), based on the t test (n = 10). BP exposure, which induced approximately 30% heart rate reduction, did not affect mRNA expression of genes related to cardiomyocyte development: hand2 (D) and nkx20.5 (E). Only BPF and BPAF significantly downregulated bmp4 mRNA expression (F), based on the one- way ANOVA test followed by Dunnett’s post hoc analysis (n = 3). SC: DMSO 0.05%, BPA: 25 µM, BPF: 60 µM, BPAF: 4 µM, BPB: 12 µM, Mix 1: BPA 6.6 µM + BPF 13.6 µM + BPAF 1 µM + BPB 3.2 µM. ns, P ≥0.03; *, P ≤0.03; **, P ≤0.002; *** , P ≤0.0002; *** *, P ≤0.0001. The data are presented as means ± standard deviations.

Article Snippet: Embryos were stained with mCherry Mouse Polyclonal Antibody (Cusabio, Houston, TX, USA), Goat Anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, and Alexa Fluor Plus 594 (Invitrogen) according to a previously described protocol (Hammond-Weinberger and ZeRuth, 2020).

Techniques: Confocal Microscopy, Expressing

Journal: Nucleic Acids Research

Article Title: Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit

doi: 10.1093/nar/gkac257

Figure Lengend Snippet: Comparisons of NCT ligands-dependent −1 PRF activity of D58 construct in different human cell-lines. ( A )The −1 PRF efficiency of D58 construct with different NCT ligands in different cell-types calculated from dual-luciferase assay results using its own in-frame control. Value for each bar is the mean of six independent experiments with standard deviation of the mean. ( B ) Fluorescent imaging analyses of HEK293T cells transfected by the D58 −1 PRF signal-containing dual-fluorescent reporter with or without ligand induction. The signals shown in the three horizontal rows were from the detection of: top panel, sfGFP (−1 frame); middle panel, mCherry (0 frame); bottom panel, bright field. ( C ) Results of Western-blot analysis of the cell lysates of dual-fluorescent reporter transfected cells in (B) using anti-mCherry (in 0 frame) or anti-GFP (in −1 frame) antibody. The cell lysates were collected after the treatment of NCT ligands for 24 h.

Article Snippet: After blocking treatment, the membrane was treated with the primary antibody mouse anti-FLAG (monoclonal antibody, 1:3000; SIGMA), mouse anti-mCherry (monoclonal antibody, 1:3000; Taiclone), chicken anti-GFP (polyclonal antibody, 1:3000; abcam) or mouse anti β-actin (monoclonal antibody, 1:5000; Proteintech), and followed by incubation with HRP-conjugated secondary antibody (anti-chicken IgG, 1:5000; Jackson or anti-mouse IgG, 1:5000; Jackson).

Techniques: Activity Assay, Construct, Luciferase, Standard Deviation, Imaging, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit

doi: 10.1093/nar/gkac257

Figure Lengend Snippet: Activation of ATF6-specific gene expression by ATF6N mimic generated from NCT-dependent −1 PRF circuit in HeLa-FS1 stable cell line. ( A ) Fluorescence microscopy images of −1 PRF circuit mediated gene expression in HeLa-FS1. The cell line was treated with tetracycline (4 μg/ml) and NCT7 (0.1 μM) or Z -NCTS (0.3 μM) for 48 h or with 2 μg/ml Tm for 1 day. The cells were fixed and stained with DAPI (upper panel), anti-Flag antibody and secondary antibody conjugated Alexa Fluor ® Plus 488 sequentially (middle panel). The images of ATF6N-induced mCherry were showed in lower panel. Bar, 10 μm. ( B ) The qRT-PCR analyses of ATF6 and XBP1s target genes in the HeLa-FS1 in (A) following expression of −1 PRF circuit and treatment with NCT7 (0.1 μM) or Z -NCTS (0.3 μM) for 24 h. The qRT-PCR values are relative to the cell line without ligand treatment, and are presented as mean with standard deviation of the mean. ** means P < 0.001 as determined by student's t -test. ( C ) The qRT-PCR analyses of ATF6 and XBP1 targeting genes of HeLa-FS1 in (A) following treatment with 10 μg/ml Tm for 6 h. The relative expression levels are calculated by comparing with those without ligand treatment after normalization, and the value for each bar is the mean with standard deviation of the mean.

Article Snippet: After blocking treatment, the membrane was treated with the primary antibody mouse anti-FLAG (monoclonal antibody, 1:3000; SIGMA), mouse anti-mCherry (monoclonal antibody, 1:3000; Taiclone), chicken anti-GFP (polyclonal antibody, 1:3000; abcam) or mouse anti β-actin (monoclonal antibody, 1:5000; Proteintech), and followed by incubation with HRP-conjugated secondary antibody (anti-chicken IgG, 1:5000; Jackson or anti-mouse IgG, 1:5000; Jackson).

Techniques: Activation Assay, Expressing, Generated, Stable Transfection, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9

doi: 10.3389/fbioe.2024.1315633

Figure Lengend Snippet: Generation of the engineered 9E10 hybridoma cell line. Flow cytometry dot plots show, after electroporation without (A) or with pX458, containing the gRNA2, and the donor construct (B) , cell viability and mCherry expression (C,D) . Negative controls (A,C) (CTR-) are reported. Data from four independent experiments are presented as mean percentage of mCherry positive cells relative to the negative control (CTR-) (E) . *** p < 0.001 (two-tailed Student’s t test).

Article Snippet: For mCherry detection, the membrane was incubated with a 1:1,000 mouse anti-mCherry monoclonal antibody (Elabscience E-AB-20087) overnight at 4°C, followed by a 1:15,000 diluition of sheep anti-mouse IgG HRP-conjugated secondary antibody (Bethyl, A90-146P) for 1 h at RT.

Techniques: Flow Cytometry, Electroporation, Construct, Expressing, Negative Control, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9

doi: 10.3389/fbioe.2024.1315633

Figure Lengend Snippet: Schematic representation of engineering of the murine IgG1 heavy-chain gene locus. The region of the wild-type (WT) mouse IgG1 heavy-chain constant gene locus involved in genome editing. The gRNA targeting the intron between the CH1 and hinge exon (H) is indicated (A) . Engineered IgH locus resulting from the HDR-based exchange of the H sequence of mouse IgG1 with that of Antarctic fish IgM (B) . The coding sequence of the insert contains the Antarctic fish hinge exon in yellow, followed by mouse IgG1 CH2 and CH3 exons, linked through a linker sequence (L) to mCherry, used as a selection marker. The stop codon is reported in white. The donor construct is flanked by the homology arms (5′HA and 3′HA, green lines) of 1,005 and 2,421 bp, respectively.

Article Snippet: For mCherry detection, the membrane was incubated with a 1:1,000 mouse anti-mCherry monoclonal antibody (Elabscience E-AB-20087) overnight at 4°C, followed by a 1:15,000 diluition of sheep anti-mouse IgG HRP-conjugated secondary antibody (Bethyl, A90-146P) for 1 h at RT.

Techniques: Sequencing, Selection, Marker, Construct

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9

doi: 10.3389/fbioe.2024.1315633

Figure Lengend Snippet: Validation of the production of anta-mAbs. Coomassie Brilliant Blue-stained 10% SDS-PAGE run under reducing conditions of purified WT and anta-mAbs. The L chain, the engineered and WT H chain bands are labeled on the right (A) . Western blot analysis using a mouse anti-mCherry monoclonal antibody as the primary antibody, followed by a sheep anti-mouse IgG HRP-conjugated secondary antibody (B) . The expected band at 75 kDa, corresponding to the engineered H chain, carrying the mCherry protein in anta-mAb, was detected. Molecular weight markers are shown on the left-hand side of each panel.

Article Snippet: For mCherry detection, the membrane was incubated with a 1:1,000 mouse anti-mCherry monoclonal antibody (Elabscience E-AB-20087) overnight at 4°C, followed by a 1:15,000 diluition of sheep anti-mouse IgG HRP-conjugated secondary antibody (Bethyl, A90-146P) for 1 h at RT.

Techniques: Biomarker Discovery, Staining, SDS Page, Purification, Labeling, Western Blot, Molecular Weight

Journal: Scientific Reports

Article Title: Considerations related to the use of short neuropeptide promoters in viral vectors targeting hypothalamic neurons

doi: 10.1038/s41598-019-47417-9

Figure Lengend Snippet: Information about all the antibodies used in this study.

Article Snippet: mouse anti-mCherry , Primary , 1/1000 , 632543 , Rockland, Limerick, PA, US , MCH-Gq.

Techniques: Concentration Assay