Journal: eLife

Article Title: Inhibition of O -GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway

doi: 10.7554/eLife.94849

Figure Lengend Snippet: ( A–D ) Real-time PCR analysis of cytokines mRNA expression in different Ogt −/− cell lines including MC38 ( A ), LLC ( B ), HT29 ( C ), B16-OVA ( D ) cells. ( E ) Western blot analysis of the activation of the interferon signaling pathway in different Ogt −/− cell lines including MC38, LLC and B16-OVA cells. ( F ) Western blot analysis of the activation of the interferon signaling pathway in Ogt −/− rescued cell lines including MC38, LLC, HT29 and B16-OVA cells. ( G–H ) Real-time PCR and western blot analysis of cytokines mRNA expression and the activation of the interferon signaling pathway in different Ogt −/− Mavs −/− double knockout clones in MC38 cells. ( I–K ) Real-time PCR and ELISA analysis of cytokines mRNA expression in Ogt −/− cGAS −/− double knockout clones in MC38 ( I–J ), HT29 ( K ) cells. ( L ) Western blot analysis of the activation of the interferon signaling pathway in Ogt −/− cGAS −/− or Ogt −/− Sting −/− double knockout clones in MC38, HT29 and B16-OVA cells. ( M–N ) BMDCs pre-treated with B16-OVA- Ogt −/− ( L ), B16-OVA- Ogt −/− cGAS −/− or B16-OVA- Ogt −/− Sting −/− cells ( M ) supernatant, and co-cultured with OT-1 T cell, then T cell proliferation was evaluated by flow cytometry, OVA 257-264 as a positive control. Representative fluorescence-activated cell sorting histograms and statistical data are shown. Data are representative of two or three independent experiments. Statistical significance was determined by unpaired Student’s t-test, one-way ANOVA, two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference. Data represent the mean of ± SD.

Article Snippet: Cell lines used in this study including B16-OVA cell line (murine melanoma, SCC-420) from Sigma-Aldrich, 293T cell line (CRL-3216), MC38 (RRID: CVCL_B288 ), LLC cell line (CRL-1642) and HT29 cell line (HTB-38) from the American Type Culture Collection.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Activation Assay, Double Knockout, Clone Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Positive Control, Fluorescence, FACS

Journal: eLife

Article Title: Inhibition of O -GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway

doi: 10.7554/eLife.94849

Figure Lengend Snippet: ( A ) The extranuclear dsDNA in different Ogt −/− MC38 cells clones were determined by PicoGreen staining assay was quantified by image J. ( B ) The extranuclear dsDNA in different Ogt −/− MC38 cells clones were determined by anti-dsDNA fluorescence staining assay and was quantified by image J. ( C ) Western blot analysis of γH2AX and H2AX expression in different Ogt −/− cell lines including MC38, LLC and B16-OVA cells. ( D ) Analysis of γH2AX and H2AX expression in different Ogt −/− clones by anti-γH2AX staining assay and was quantified by image (J). ( E ) The DNA damage was determined by comet assay, and extranuclear dsDNA was analyzed by using CometScore in Ogt −/− MC38 cells. ( F ) Western blot analysis of γH2AX and H2AX expression in Ogt −/− rescued cells including MC38, LLC, HT29 and B16-OVA cells. ( G ) The DNA damage in rescued MC38 cells were determined by comet assay, and extranuclear dsDNA was analyzed by using CometScore. Data are representative of three or four independent experiments. Statistical significance was determined by unpaired Student’s t-test, one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference. Data represent the mean of ± SD.

Article Snippet: Cell lines used in this study including B16-OVA cell line (murine melanoma, SCC-420) from Sigma-Aldrich, 293T cell line (CRL-3216), MC38 (RRID: CVCL_B288 ), LLC cell line (CRL-1642) and HT29 cell line (HTB-38) from the American Type Culture Collection.

Techniques: Clone Assay, Staining, Fluorescence, Western Blot, Expressing, Single Cell Gel Electrophoresis

Journal: eLife

Article Title: Inhibition of O -GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway

doi: 10.7554/eLife.94849

Figure Lengend Snippet: ( A ) Volcano plot of OGT binding proteins identified by LC–MS/MS from stably expressed exogenous GFP-OGT in OGT knockout HT29 cells. ( B ) OGT and HCF1 binding was confirmed by immunoprecipitation assay in OGT rescued HT29 cells. ( C ) OGT and HCF1 binding was confirmed by immunoprecipitation assay in 293T cells. ( D ) HCF1 cleavage was confirmed by western blot in Ogt rescued MC38 cells. ( E ) Co-IP analysis of the interaction between OGT and different HCF-1 mutant. ( F ) Real-time PCR analysis of cytokines mRNA expression effected by HCF-1 isoforms in MC38 OGT knockout cells. ( G ) Western blot analysis of γH2AX and H2AX expression in exogenous HCF-1 C600 expressed MC38 Ogt knockout cells. ( H ) The extranuclear dsDNA were determined by anti-dsDNA fluorescence staining assay and was quantified by image J in exogenous HCF-1 C600 expressed MC38 OGT knockout cells. Data are representative of three or four independent experiments. Statistical significance was determined by one-way ANOVA, two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference. Data represent the mean of ± SD.

Article Snippet: Cell lines used in this study including B16-OVA cell line (murine melanoma, SCC-420) from Sigma-Aldrich, 293T cell line (CRL-3216), MC38 (RRID: CVCL_B288 ), LLC cell line (CRL-1642) and HT29 cell line (HTB-38) from the American Type Culture Collection.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Knock-Out, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, Fluorescence, Staining

Journal: Scientific Reports

Article Title: Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions

doi: 10.1038/s41598-022-18400-8

Figure Lengend Snippet: The differentiation potential of cells in culture can be altered by culture medium. ( A ) H9 cells cultured with Essential 8 (Es8) medium on vitronectin-N (VTN)–coated dishes were transferred to RFF2 medium, cultured for 15 days (3 days/passage × 5 passages), transferred again to Es8 medium, cultured 24 days (3 days/passage × 8 passages), and then transferred again to RFF2 medium. Photos of cells in designated culture conditions, with the cell number scored at day 3 after seeding 1.0 × 10 5 cells (left panels); flow cytometric analysis of CHD7, CHD7 copy numbers from 5 ng total RNA at day 3 (middle panels); and photographs of EBs formed by day 14 from cells in each culture condition and numbers of EBs formed (right panels). The results are representative of three independent experiments. ( B ) H9 cells were cultured either with Es8 or RFF2 on VTN-N–coated dishes. The loci of copy number variants (CNVs) detected when cells were cultured with Es8 medium (left panels) or RFF2 medium (right panels) are shown. CHD7 expression was determined by flow cytometry (mean values are shown), and CHD7 copy numbers were determined by digital droplet PCR in cells cultured with Es8 or RFF2 medium.

Article Snippet: Briefly, cDNA was synthesized from 5 ng total RNA extracted from cells cultured with hPSCs or Es8 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Cell Culture, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions

doi: 10.1038/s41598-022-18400-8

Figure Lengend Snippet: Activation of mitochondrial function is coupled with differentiation. ( A ) Morphology, CellROX (ROX) immunostaining, CHD7 copy numbers, and mitochondrial membrane voltage (JC-1 assays) in cells cultured with Es8 medium on VTN-N–coated dishes (Es8/VTN) for 3 days (left panels) or with RFF2 medium on VTN-N–coated dishes (RFF2/VTN) for 3 days (right panels) are shown. Mitochondrial membrane voltage was assessed by subtracting baseline electrons (after depolarization) from total electrons (red circle). The percentage of each fraction in the scatter plot of JC-1 assays is shown. ( B ) H9 cells were cultured with Es8 or RFF2 medium, and culture medium was collected and replaced with fresh medium every day for 3 days. 2-Aminoadipic acid (2-AAA), malate, and citrate levels in culture medium were measured using LC–MS/MS. The measured values were standardized as the mean area ratio/cell/h for 3 days. The average values (n = 3) with error bars (SD) are shown in the bar graphs. The results of three independent experiments are shown. ( C ) Morphology, ROX staining, mitochondrial membrane voltage (JC-1 assays; red circle), and gene expression profiles (RT-qPCR score card panels) of H9 cells cultured with Es8 medium on VTN-N–coated dishes on day 5 (left panel: starting material for differentiation by Es6 medium) and Es6 medium on VTN-N–coated dishes on day 5 are shown (right panel). The interpretation of gene expression levels by RT-qPCR is shown in the attached table. The results of three independent experiments are shown.

Article Snippet: Briefly, cDNA was synthesized from 5 ng total RNA extracted from cells cultured with hPSCs or Es8 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Activation Assay, Immunostaining, Membrane, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Staining, Gene Expression, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions

doi: 10.1038/s41598-022-18400-8

Figure Lengend Snippet: CHD7 expression affected the differentiation potential and growth of undifferentiated cells. ( A ) H9 cells cultured with Es8 on VTN-N–coated dishes (Es8/VTN, left panels) or with RFF2 on VTN-N–coated dishes (RFF2/VTN, right panels) were transfected with mock (control), mCHD7 , or siCHD7 . The morphology, CHD7 copy numbers, gene expression profiles (RT-qPCR), EB morphology, and EB numbers formed at day 14 under different culture conditions are shown. The representative results of three independent experiments are shown. ( B ) CHD7 expression in H9 cells determined by flow cytometry after cells were transferred from RFF2 to Es8 on VTN-N–coated dishes at passage 0 (P0), P5, and P7. Cells were cultured for 3 days between passages. ( C ) Fold increase of H9 cells after 48 h (upper panel) and CHD7 expression, as determined by RT-qPCR, after transfection of H9 cells with various doses of siCHD7 (lower panel). The average values (n = 3) with error bars (SD) are shown in the bar graphs. Representative data from three independent experiments are shown.

Article Snippet: Briefly, cDNA was synthesized from 5 ng total RNA extracted from cells cultured with hPSCs or Es8 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Expressing, Cell Culture, Transfection, Control, Gene Expression, Quantitative RT-PCR, Flow Cytometry

Journal: Scientific Reports

Article Title: Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions

doi: 10.1038/s41598-022-18400-8

Figure Lengend Snippet: Recloning of cells with differentiation potential based on culture conditions. ( A ) iPSC clones (201B7, PFX#9, SHh#2, or 253G1 cells) or ESC clones (H9 cells) were cultured either on feeder cells or on L511- or L521-coated dishes with iPSC or mTeSR1 medium. Clones were then transferred to Es8 medium and cultured on VTN-N–coated dishes. The mean and convergence of CHD7 expression of cell clones was determined by flow cytometry before (gray histogram) and after (red histogram) changing culture conditions. Representative results from three independent experiments are shown. ( B ) Flow cytometric analysis of cell clones for the mean and coefficient of variation (CV) measured before (circle) and after (square) changing culture conditions are plotted on the left panel and the differentiation potential before and after changing the culture conditions was assessed by the number of EBs formed and is shown on the right panel. The data set shown in ( B ) was generated from the same samples shown in ( A ).

Article Snippet: Briefly, cDNA was synthesized from 5 ng total RNA extracted from cells cultured with hPSCs or Es8 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Clone Assay, Cell Culture, Expressing, Flow Cytometry, Generated

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: DTX3L and USP28 interact with each other (A) Interaction of endogenous DTX3L and USP28 was visualized by proximity ligation assay (PLA) in SK-MES-1 cells. Cells were grown on cover slips, fixed and immunostained according to Duolink manufacture’s protocol. Red spots reflect the interaction. DAPI stained nuclei are shown in blue. Scale bars: A-A‴ 50 μm and a-a’’’ 20 μm. (B) Quantification of PLA signals. ∗∗∗∗significant difference between the number of proximity ligation sites in the negative control vs. DTX3L/USP28. Data are mean ± SD from 3 independent experiments. The statistical significance of differences was determined by ordinary one-way ANOVA. ∗∗∗ p < 0.001. (C and D) Cell extracts from PC-3 cells were immunoprecipitated with either IgG control or USP28 antibodies. Resulting precipitates were first analyzed by immunoblotting (IB) using a DTX3L; thereafter, the membrane was re-probed with a USP28 antibody. Whole cell lysate (C) and cytoplasmic (CF) and nuclear fraction (NF) (D). (E) HEK293 cells were cotransfected with expression vectors encoding full length Flag-tagged USP28 and full-length HA-tagged DTX3L or the N-terminal HA-tagged DTX3L (A2-A516) deletion mutant or the C-terminal HA-tagged DTX3L (K555-E740) deletion mutant. Cell extracts were immunoprecipitated with either IgG control or USP28 antibodies. Immunoprecipitates were analyzed by immunoblotting (IB) using an HA-tag antibody. (F) Representative MST binding curve for DTX3L and USP28 interaction. All binding curves are shown in .

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Proximity Ligation Assay, Staining, Ligation, Negative Control, Immunoprecipitation, Control, Western Blot, Membrane, Expressing, Mutagenesis, Binding Assay

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: Functional interplay between USP28 and DTX3L (A) In vitro ubiquitination assay indicating the auto-ubiquitination activity of DTX3L and deubiquitinating activity of USP28. Protein concentrations used were Ub (50 μM), E1 (0.4 μM), UbcH5a (E2) (2 μM) and DTX3L (0.7 μM). USP28 was used at a final concentration of 3 μM. SDS-PAGE showing appearance of the DTXL3 ubiquitination pattern that is visible as a smear (lane 8) and its disappearance due to hydrolysis by USP28 (lane 9) and/or the USP28 catalytic domain (lane 11). Ubiquitinated DTX3L appears as a high molecular weight smear and the black arrow indicates a band that corresponds to ubiquitinated USP28. Note, that for unknown reasons USP28 preparations showed some degradation (lanes 5–6). (B) Western blot of the assay in panel A probed with an anti-ubiquitin antibody. The black arrow indicates a band that corresponds to ubiquitinated USP28. (C) Western blot of the assay in panel A probed with anti-USP28. The arrow indicates a band shift of the ubiquitinated catalytically inactive mutant USP28 C171A in the presence of ATP, Ubiquitin (Ub), E1 and E2 ligases as well as DTX3L. (D–H) Cell based ubiquitination assays. HEK293 cells were transfected with an empty vector (Ctl) or expression vectors for DTX3L, wild type USP28 and the catalytically inactive mutant USP28 C171A . In D, ubiquitinated DTX3L was detected with ubiquitin antibody after immunoprecipitation of DTX3L. In (E), ubiquitinated USP28 was detected with ubiquitin antibody after immunoprecipitation of USP28 with a FlagM2-tag antibody. In (F), cells were transfected with DTX3L and Flag-USP28 expression vectors as well as with empty vector (Ctl) or vectors for wild type ubiquitin (Ub), a mutant in which all lysine residues were converted to arginine (Ub-KR) or mutants where either only K48 (Ub-K48) or K63 (Ub-K63) remained intact. Ubiquitinated USP28 was detected with ubiquitin antibody after immunoprecipitation of USP28 with a FlagM2-tag antibody. In (H), cells were transfected with HA-DTX3L and Flag-USP28 expression vectors as well as with empty vector (Ctl) or vectors for wild type ubiquitin and the different ubiquitin mutants as in (F). Ubiquitinated DTX3L was detected with ubiquitin antibody after immunoprecipitation of DTX3L with an HA-tag antibody. (G) Domain organization of USP28 and the ubiquitination sites identified by mass spectrometry using the USP28 C171A mutant. UBA - ubiquitin-associated domain; UIM -ubiquitin interaction motif; SIM - SUMO interaction motif.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Functional Assay, In Vitro, Ubiquitin Proteomics, Activity Assay, Concentration Assay, SDS Page, High Molecular Weight, Western Blot, Electrophoretic Mobility Shift Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Mass Spectrometry

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: Mutual regulation of DTX3L and USP28 protein levels (A) SK-MES-1 cells were transfected either with expression vector for scrambled control shRNA (shScr), or with expression vectors for full-length DTX3L or one of two independent shRNAs (shDTX3L_8, shDTX3L_11) against DTX3L. After transfection, cells were further cultured under normoxia (16% O 2 ) or hypoxia (5% O 2 ) for 4 h. (B) USP28, HIF-1α, p53, c-MYC, and DTX3L protein levels were measured by Western blot analysis. Alpha tubulin served as a loading control. (C) SK-MES-1 cells were treated with the USP28/25 inhibitor AZ1 (10 μM) and cultured under normoxia and hypoxia for 4 h and 24 h. (D) DTX3L, HIF-1α, p53, and c-MYC protein levels were measured by Western blot analysis. (E) SK-MES-1 cells were transfected with expression vectors for scrambled control shRNA (shScr) or shRNA 1 or shRNA 3 against USP28. After transfection, cells were further cultured under normoxia or hypoxia for 4 h. DTX3L, HIF-1α, p53, c-Myc and USP28 protein levels were measured by Western blot analysis. (B–F) Quantification of USP28, DTX3L, HIF-1α, p53 and c-MYC. In each experiment the protein levels at 5% O 2 control (Ctl) or shScr were set to 100%. ∗significant difference for 16% O 2 : Ctl vs. DTX3L, vs. shDTX3L, vs. shUSP28 or vs. AZ1, § significant difference between 16% O 2 vs. 5% O 2 , # significant difference for 5% O 2 : Ctl vs. DTX3L, vs. shDTX3L, vs. shUSP28 or vs. AZ1, p < 0.05.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, shRNA, Cell Culture, Western Blot

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: DTX3L and USP28 control each other’s half-life (A and C) HEK293 cells were transfected with scrambled shRNA or with two different shRNA’s against either DTX3L or USP28. After transfection, protein synthesis was inhibited with cycloheximide (CHX; 10 μg/μL) and cells were harvested at indicated time points. In each experiment the protein levels at time point 0 were set to 100%. ∗significant differences shScr vs. shDTX3L or shUSP28_1, # significant differences shScr vs. shDTX3L_11 or shUSP28_3, p ≤ 0.05. (B and D) Representative Western blot analysis. 100 μg of total protein lysate was analyzed with antibodies against USP28, DTX3L and α-tubulin.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Control, Transfection, shRNA, Western Blot

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: NHEJ-, and HR-DSB repair analysis (A) Induction of DSBs by bleomycin (BLM) does not affect USP28/DTX3L interaction. Cells treated with BLM (10 μM) and the proteasome inhibitor MG132 were immunoprecipitated with either IgG control or USP28 antibodies. Resulting precipitates were analyzed by immunoblotting (IB) using a DTX3L antibody. Membrane was re-probed with an antibody against USP28. (B and E) Scheme of pathway-specific repair activities for NHEJ and HR measured with EGFP reporter assays. (C and F). Quantification of NHEJ and HR DSB repair activities in BLM treated MDA-MB-231 Scr, USP28-KD, DTX3L-KD and USP28/DTX3L double knockdown cells. Mean values for the MDA-MB-231 Scr cells (shScr) were defined as 100%. Data are mean ± SD from 6 measurements. The statistical significance of differences was determined using ordinary one-way ANOVA. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (D and G) Representative flow cytometry images.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Immunoprecipitation, Control, Western Blot, Membrane, Knockdown, Flow Cytometry

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: SSA-, and MMEJ-DSB repair analysis (A and D) Scheme of pathway-specific repair activities for NHEJ and HR measured with EGFP reporter assays. (B and E). Quantification of NHEJ and HR DSB repair activities in BLM treated MDA-MB-231 Scr, USP28-KD, DTX3L-KD and USP28/DTX3L double knockdown cells. Mean values for the MDA-MB-231 Scr cells (shScr) were defined as 100%. Data are mean ± SD from 6 measurements. The statistical significance of differences was determined using ordinary one-way ANOVA. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C and F) Representative flow cytometry images. (G) Representative images of Comet assay. Scale bar: 1000 μM. (H) Determination of % of DNA in tail in MDA-MB-231 cells transfected with the indicated shRNAs. Data were expressed as mean ± SD of three independent experiments ( n > 50 cells per condition). Statistical significance was determined using ordinary one-way ANOVA. ∗∗∗∗ p < 0.001. (I) Representative immunofluorescence images of γH2AX (green) and HOECHST nuclear staining (blue). Scale bar:50 μm. (J) Quantification of mean γH2AX foci per nucleus in MDA-MB-231 cells transfected with the indicated shRNAs. Data are mean ± SD from three experiments ( n > 100 cells per condition). Statistical significance was determined using ordinary one-way ANOVA. ∗∗∗∗ p < 0.001.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Knockdown, Flow Cytometry, Single Cell Gel Electrophoresis, Transfection, Immunofluorescence, Staining

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: Mutual regulation of cell cycle and apoptosis by USP28 and DTX3L upon DNA DSB induction. MDA-MB-231 Scr, USP28-KD, DTX3L-KD and USP28/DTX3L double knockdown cells were treated with bleomycin (BLM; 10 μM) and further cultured for 24 h (A) Cell cycle distribution of MDA-MB-231 shScr, USP28 KD, DTX3L KD, and double knockdown (USP28 KD + DTX3L KD) cells in the G1/0 phase, S phase, and G2 phase. Significance ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (B) Quantification of apoptosis in MDA-MB-231 Scr, USP28 KD, DTX3L KD and USP28/DTX3L double knockdown cells Significance ∗ p < 0.05, ∗∗ p < 0.01. (C) Histograms of apoptotic cells assessed by Annexin-V/PI staining and measured by flow cytometry.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Knockdown, Cell Culture, Staining, Flow Cytometry

Journal: Bioinformatics advances

Article Title: Different approaches to Imaging Mass Cytometry data analysis.

doi: 10.1093/bioadv/vbad046

Figure Lengend Snippet: Fig. 2. A schematic overview of the Imaging Mass Cytometry workflow (created with BioRender.com)

Article Snippet: IMC was developed in 2014 based on earlier available suspensionbased, single-cell mass cytometry [cytometry time of flight (CyTOF)] technology [described in Bandura et al. (2009)] but combined with an additional platform for UV ablation (Hyperion Tissue Imager, Standard BioTools, South San Francisco, USA) and performed on stained tissue sections, giving the spatial resolution of the data (Fig. 1A) (Bandura et al., 2009).

Techniques: Imaging, Mass Cytometry

Journal: bioRxiv

Article Title: O-GlcNAcylation and low glycolysis underpin Th2 polarization by dendritic cells

doi: 10.64898/2026.02.03.703465

Figure Lengend Snippet: a, Schematic of the hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation. b, Relative mRNA expression of 24h-conditioned human moDCs based on bulk RNA sequencing data . c , Relative intracellular metabolite abundance of 24h-conditioned human moDCs based on flow injection analysis mass spectrometry (FIA-MS; ). d, Volcano plot showing differences in intracelullar metabolite abundance between Th2-DCs and iDCs. Positive values corresponding to metabolites with increased abundance in Th2-DCs. Significantly different metabolites are highlighted in red. e , Integration of metabolomic and transcriptomic data sets using CoMBI-T profiling related to the HBP pathway (as described in ). Round nodes represent metabolites within the core regulatory network. Enzymes are represented by edges. Differential expression of enzymes or abundance of metabolites are indicated by red (higher in Th2-DCs) to green (higher in iDCs) color scale. f, Flow cytometry-based analysis of overall protein O-GlcNAcylation of conditioned human moDCs. g, i-k, Human moDCs were preincubated with inhibitors of O-GlcNAcylation (ST045849 – OGTi), or N-glycosylation (tunicamycin) for 30 minutes, before stimulation with g , omega-1, i , House dust mite (HDM) extract, 2-DG, j , LPS/Poly-IC or Zymosan, after which washed DCs were cocultured with ( g-j ) allogeneic naïve Th cells and 11 days later Th1/2 cell polarization was determined by intracellular cytokine staining or k , with memory Th cells and 5 days after DC-T cell coculture and IL-17 concentrations were determined by ELISA. ( G ) Representative flow plots are shown on the left. On the right % cytokine-producing T cells are shown relative to control conditioned-DCs. h, Differentiating moDCs were transduced with short interfering RNA against O-GlcNAc transferase (OGT) at day 4 and subsequently analyzed as in ( g ). Scrambled RNA was used as a control. Data points represent independent experiments of individual donors. Bars represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. Statistical significance was determined using one-way ANOVA with Dunnet post-hoc test ( f,g,i-k ) and Student’s t -test ( h ).

Article Snippet: Inhibitors included: 5-10 mM of 2-deoxyglucose (2-DG; in mQ water; #D8375-1g, Sigma), 20 μM ST045849 (ST; in DMSO; #6775, Tocris) and 10 μM Thiamet G (TG; in DMSO; #13237, Cayman Chemical, Ann Arbor, Michigan, United States).

Techniques: Expressing, RNA Sequencing, Injection, Mass Spectrometry, Metabolomic, Quantitative Proteomics, Flow Cytometry, Glycoproteomics, Staining, Enzyme-linked Immunosorbent Assay, Control, Transduction, Small Interfering RNA

Journal: bioRxiv

Article Title: O-GlcNAcylation and low glycolysis underpin Th2 polarization by dendritic cells

doi: 10.64898/2026.02.03.703465

Figure Lengend Snippet: ( A ) MS/MS based proteomic analysis of O-GlcNAcylated proteins in differently conditioned human moDCs. Volcano plot showing differential protein O-GlcNAcylation in Th2-DCs compared with iDCs. ( B ) Protein–protein interaction network of proteins exhibiting increased or decreased O-GlcNAcylation in Th2-DCs. ( C ) Gene Ontology (GO) enrichment analysis of cellular component terms associated with differentially O-GlcNAcylated proteins in Th2-DCs. ( D ) Confocal microscopy of F-Actin (green), Zyxin (red), and merged images of moDCs left untreated or stimulated for 24 h with LPS, omega1, or omega1 preincubated with the O-GlcNAcylation inhibitor ST045849 (OGTi) for 30 minutes. ( E ) quantification of the percentage of overlap between F-Actin and ZYXIN in individual cells. ( F ) Conditioned human moDCs were co-cultured with allogenic naïve Th cells for 4h. DC-T cell conjugates were identified as CD1a + CD4 + dual-positive events and quantified as the percentage of total DCs. ( G ) A schematic diagram for Atomic Force Microscopy (AFM)-based single-cell force spectroscopy (SCFS) assay setup. ( H ) DC2.4 cells were stimulated with LPS or omega1 with or without pre-incubated with OGTi prior to stimulation. Adhesion was quantified as in (G). ( I ) WT, Fascin ko or Zxyin ko DC2.4 cells were stimulated with LPS or omega1 with or without pre-incubated with OGTi prior to stimulation. Adhesion assay following knockout as in (G) ( J ) siRNA mediated knockdown of Fascin-1 or Zyxin was performed on day 4 of moDCs differentiation; scrambled siRNA served as control. On day 6, DCs were co-cultured with naïve allogeneic T cells for 11 days. Intracellular IFN-γ and IL-4 production was assessed by flow cytometry after 6h of PMA/ionomycin stimulation. Percentages of Th cells expressing IFN-γ or IL-4 are shown relative to untreated scrambled control. ( A-C ) Analysis based on data from 4 donors. One representative of ( D ) 4 or ( H,I ) 2 independent experiments is shown. ( F,J ) Datapoints represent individual donors from independent experiments. Data are shown as mean ± SEM. Statistical significance was determined by (E) paired Student’s t-test or (G-I) using two-way ANOVA with Tukey post-hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Inhibitors included: 5-10 mM of 2-deoxyglucose (2-DG; in mQ water; #D8375-1g, Sigma), 20 μM ST045849 (ST; in DMSO; #6775, Tocris) and 10 μM Thiamet G (TG; in DMSO; #13237, Cayman Chemical, Ann Arbor, Michigan, United States).

Techniques: Tandem Mass Spectroscopy, Confocal Microscopy, Cell Culture, Microscopy, Single Cell, Force Spectroscopy, Incubation, Cell Adhesion Assay, Knock-Out, Knockdown, Control, Flow Cytometry, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of fetal microchimeric immune cells in mouse maternal hearts during physiologic and pathologic pregnancies

doi: 10.3389/fcell.2023.1256945

Figure Lengend Snippet: Methodology framework for high-dimensional mass cytometry. (A) Male homozygous mT/mG +/+ C57BL/6J mice were mated with wild-type females, which resulted in progeny cells that express mT+. Pregnant mice were treated intravaginally with 10 4 CFU E. coli to mimic ascending infection, with sterile nutrient broth serving as negative control. (B) Both flow cytometry and mass cytometry utilize cell labeling with antibodies to facilitate single-cell analysis, although fewer spectral overlap is observed with mass cytometry. (C) Molecules of interest were tagged with elemental isotope-labelled antibodies. Following nebulization and injection into a mass analyzer, the ionized metals were accelerated through a vacuum chamber and were analyzed based on time of flight.

Article Snippet: A day prior to experiment, a bacterial culture of ATCC 12014 Escherichia coli O55:K59(B5):H– (Lot #496291, ThermoFisher Scientific Remel Products, San Diego, CA, United States) was inoculated in 200 mL sterile nutrient broth (Difco, Cat. # 234000, BD Biosciences, Franklin Lakes, NJ, United States) and cultured for 16 h at 37°C with shaking.

Techniques: Mass Cytometry, Infection, Sterility, Negative Control, Flow Cytometry, Labeling, Single-cell Analysis, Injection

Journal: Cell reports

Article Title: PPARγ Interaction with UBR5/ATMIN Promotes DNA Repairto Maintain Endothelial Homeostasis

doi: 10.1016/j.celrep.2019.01.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: UBR5/EDD1 Antibody , Bethyl , Cat#A300-573A, RRID:AB_2210189.

Techniques: Control, Recombinant, In Vitro, Transfection, Flow Cytometry, Single Cell Gel Electrophoresis, Mass Spectrometry, Sequencing, Real-time Polymerase Chain Reaction, Cloning, Plasmid Preparation, Software

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Structure–function analysis of BCL11A defines a region of BCL11A that interacts with NTHL1 and stimulates its enzymatic activity. ( A ) Diagrammatic representation of the BCL11A-XL and fragments tested in pull-down and DNA repair assays. ( B ) Diagrammatic representation of the 5,6-dihydrothymidine (DHT) cleavage assay using a fluorophore reporter probe. The assay was performed using 5 nM of NTHL1 and 20 nM of BCL11A-XL, in the presence of 50 nM of BSA. ( C ) Diagrammatic representation of the thymine glycol (Tg) cleavage assay using a radioactively labeled probe. The assay was performed using purified NTHL1 (10 nM) and the indicated amounts of BSA and/or BCL11A-XL. The reaction in lane 1 was performed with a probe that contains a normal thymidine base instead of thymine glycol. ( D ) Pull-down assays were performed using His-tagged BCL11A fragments and either GST or GST-NTHL1, followed by immunoblotting with anti-His antibodies. ( E ) DHT cleavage assays were performed using 20 nM of BCL11A fragments, in the presence of absence of NTHL1 (5 nM), as indicated. An additional assay was performed with 40 nM of BCL11A 160–520 . All samples included 50 nM of BSA; the sample labeled ‘BSA’ included an additional 20 nM BSA. ( F ) Thymine-glycol (Tg) cleavage assays were performed using radioactively end-labeled double-stranded oligonucleotides containing a Tg oxidized base, and 250 nM BSA. Where indicated, NTHL1 (2.5 nM), BSA or BCL11A 160–520 (15 nM) were included in the reaction. Production of a cleaved product in samples that have not been treated with NaOH (lanes 1–4) requires the glycosylase and AP/lyase activities of NTHL1. In the absence of NaOH, NTHL1 alone cleaves 9.7% of the substrate, whereas BCL11A 160–520 cleaves 49%. In the presence of NaOH, NTHL1 alone cleaves 20.3% of the substrate, whereas BCL11A 160–520 cleaves 43.8%. ( G ) Radioactively end-labeled double-stranded oligonucleotides containing a thymine glycol (Tg) base were incubated with the indicated amount of NTHL1, BSA and BCL11A 160–520 . After incubation at 37°C, 50 mM sodium borohydride was added. The reactions were incubated for another 15 min at 37°C. After termination of the reaction, the trapped complexes were separated from free DNA by 10% SDS-PAGE. ( H ) Pull-down assays were performed using the His-tagged BCL11A 161-366 peptide and either GST or GST-NTHL1 1-312 , GST-NTHL1 88-312 or GST-NTHL1 1-116 followed by immunoblotting with anti-His antibodies.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Activity Assay, Cleavage Assay, Labeling, Purification, Western Blot, Incubation, SDS Page

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: The DNA repair and proliferation defects caused by BCL11A knockdown are rescued by ectopic expression of the BCL11A 160–520 Fragment. ( A , B , E and F ) MDA-MB-231 cells were infected with lentiviral vectors as indicated: an empty vector, a vector expressing BCL11A shRNA, and a vector expressing BCL11A 160–520 . ( A ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( B ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH 10 after treatment of cells with the Endo III DNA glycosylase. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( C ) MDA-MB-231 cells stably carrying an empty vector or a vector expressing BCL11A 160–520 were transfected with either of two distinct BCL11A dicer RNAs or a control dicer RNA. Genomic DNA was purified and abasic sites were quantified using an aldehyde-reactive probe. Results are an average of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( D ) MDA-MB-231 cells were stably infected with a vector expressing BCL11A shRNA under the control of a tetracycline-inducible promoter, as well as an empty vector or a vector expressing BCL11A 160–520 . Cells were treated or not with doxycycline for 3 days before cell extracts were prepared and analyzed in a DNA repair assay using as a substrate double-stranded oligonucleotide containing a thymine glycol base (Tg). Upon incubation in the presence of 32 P-dTTP, NTHL1 present in the cell extract removes the thymine glycol and introduces a single-strand break; Pol β adds a radioactively labeled thymidine and ligase III seals the strand-break thereby generating a single-strand that migrates more slowly on a denaturing gel. The amount of repair completion (top band) for each sample was normalized to the value of ‘Vector no dox’. The enzymatic activity of the BCL11A knockdown (‘vector + dox’) is 55.7% whereas the activity of ‘BCL11A 160–520 no dox’ is 365% and ‘BCL11A 160–520 + dox’ is 361%. ( E ) Cells were treated with increasing amounts of H 2 O 2 and then submitted to a clonogenic assay. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( F ) MDA-MB-231 cells carrying BCL11A 160–520 or not were infected with lentiviruses expressing either control or BCL11A shRNA. β-Gal associated senescence was measured 5 days after infection. All values are normalized to the control shRNA value. Error bars represent standard error. Results are a representative of one of two different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( G ) MDA-MB-231 cells were stably infected with a lentiviral vector expressing a BCL11A shRNA under the control of a doxycycline-inducible promoter and either an empty vector or a vector expressing BCL11A 160–520 . Doxycycline was added to the medium or not and 3 days later cell proliferation was measured by staining with CellTrace™ CFSE. CFSE was added to the medium and a portion of the population was fixed immediately as the ‘0’ generation. The remaining cells were allowed to proliferate for 3 days. Cells were fixed and analyzed by flow cytometry. Small peaks within the CFSE profiles represent successive generations, as indicated above the peaks. The proliferation index is the total number of divisions divided by the number of cells that went into division. The division index is the average number of cell divisions, taking into account the cells that never divided.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Knockdown, Expressing, Infection, Plasmid Preparation, shRNA, Single Cell Gel Electrophoresis, Stable Transfection, Transfection, Control, Purification, Incubation, Labeling, Activity Assay, Clonogenic Assay, Staining, Flow Cytometry

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A interacts with Pol β and stimulates its enzymatic activities. ( A ) Nuclear protein extracts (500 μg) from MDA-MB-231 cells were submitted to co-immunoprecipitation (IP) alternatively with Pol β or BCL11A antibodies and then immunoblotted (IB) with either anti-Polβ or anti-BCL11A antibody. Input (1%) was loaded as a protein expression control. ( B ) 293 cells were transfected with vectors expressing fusion proteins containing either the N-terminal or C-terminal portion of Intein, as indicated: BCL11A 160–520 -V5-IN4b with either IC2-Flag-LIG3, IC2-Flag-FOXN2 or IC2-Flag-Pol β. Whole cell extracts were submitted to immunoblotting analysis with the V5 and FLAG antibodies. ( C ) Pull-down assays were performed using His-tagged BCL11A fragments and either GST or GST-Pol β, followed by immunoblotting with anti-His antibodies. ( D ) Diagrammatic representation of the strand displacement assay using a fluorophore reporter probe. F: FAM fluorophore; Q: quencher. The strand displacement assay was performed using 2.5 nM of BCL11A and 15 nM of Pol β. 50 nM of BSA were added to each reaction. ( E ) Double-stranded oligonucleotides containing a uracil residue were incubated with UDG and APE1 to form a gapped substrate for Pol β. The DNA was then incubated with 32 P-dCTP in the presence of DNA Pol β and either BSA or BCL11A 160–520 . ( F ) Double-stranded oligonucleotides containing a uracil residue were labeled at the 5'-end and incubated with UDG and APE1 to form a gapped substrate for Pol β. The gapped probe was then incubated with all 4 dNTPs in the presence of DNA Pol β and either 25 nM BSA or 25 nM BCL11A 160–520 . ( G ) Double-stranded oligonucleotides containing a uracil residue, that were labeled at the 3′ end using Klenow and fluorescent CF 660R dCTP, were incubated with UDG and APE1 to produce a single-strand nick with a 5′-deoxyribose phosphate (dRP). This DNA substrate was incubated with Pol β in the presence of BSA or BCL11A 160–520 . NaBH4 was added to most samples, except in lane 9, to prevent the spontaneous conversion of dRP into P. From four independent pairs of assays, we calculated an average 1.8 fold (±0.3) stimulation of Pol β dRP-lyase activity by BCL11A 160–520 .

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Immunoprecipitation, Expressing, Control, Transfection, Western Blot, Residue, Incubation, Labeling, Activity Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: The impact of BCL11A knockdown on DNA repair, genomic DNA damage and clonogenic efficiency is confirmed in other triple-negative breast cancer cells. (A and B) The triple-negative breast cancer cell lines BT549, MDA-MB-468 and Hs578T were transfected with either of two distinct BCL11A dicer RNAs or a control dicer RNA. After 2 days, cell extracts were prepared and in parallel cells were submitted to a comet assay. ( A ) Cell extracts were prepared and analyzed in a DNA repair assay with a probe containing a thymine glycol, as described in Figure . ( B ) A fraction of the cells was submitted to single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) Cells were stably infected with a lentiviral vector expressing a BCL11A shRNA under the control of a doxycycline-inducible promoter. Doxycycline was added to the medium or not and 3 days later cells were treated with the indicated concentrations (0, 20, 50) of H 2 O 2 and then submitted to a clonogenic assay. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Knockdown, Transfection, Control, Single Cell Gel Electrophoresis, Stable Transfection, Infection, Plasmid Preparation, Expressing, shRNA, Clonogenic Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Proximity Biotinylation with NTHL1-BirA*. ( A ) Flow chart of manipulations for the BioID experiment. Following treatment with tetracycline and addition of biotin to the medium, cells were treated (2 Gy) or not (0 Gy) with ionizing radiation prior to cell lysis, affinity purification on streptavidin beads and mass spectrometry identification. ( B ) Immunoblotting analysis of Flp-In™ T-Rex™ 293 cells that were engineered to stably carry vectors that express BirA*-FLAG or NTHL1-BirA*-FLAG upon tetracycline induction. ( C ) Immunoblotting analysis with streptavidin confirming the expression of BirA* fusion proteins and successful biotinylation by BirA*. ( D ) The Venn diagram shows the number of NTHL1-BirA* target proteins identified from mass spectrometry after removing all candidates with BFDR above 0.2, and proteins identified in cells expressing BirA*-FLAG and NLS-BirA*-FLAG. 146 preys were identified in both unirradiated and irradiated cells, while 41 and 36 preys were respectively identified only in unirradiated or irradiated cells. ( E ) Nuclear protein extracts (500 μg) from MDA-MB-231cells were submitted to immunoprecipitation (IP) alternatively with NTHL1 or BCL11A antibodies and then immunoblotted (IB) with either anti-NTHL1 or anti-BCL11A antibody. The control lanes did not include a primary antibody while the input was loaded directly onto the gel. ( F ) 293 cells were transfected with vectors expressing BCL11A and NTHL1 fusion proteins containing the N-terminal or C-terminal portion of intein, respectively: BCL11A-V5-IN and IC2-FLAG-NTHL1. Whole cell extracts were submitted to immunoblotting analysis with the V5 and FLAG antibodies. The new band detected by both antibodies represents the recombined protein.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Lysis, Affinity Purification, Mass Spectrometry, Western Blot, Stable Transfection, Expressing, Irradiation, Immunoprecipitation, Control, Transfection

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A knockdown causes an increase in genomic DNA damage and a delay in the repair of oxidized bases and abasic sites. MDA-MB-231 cells were infected with lentiviral vectors expressing a BCL11A shRNA or a non-targeting sequence. Knockdown of BCL11A is ∼60%. ( A ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated times before carrying out single cell gel electrophoresis at pH > 13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( B ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH 10 and pH 10 after treatment of cells with the Endo III DNA glycosylase. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) Cells stably carrying a lentivirus expressing an shRNA against BCL11A or an empty vector were treated or not with 50 μM H 2 O 2 . Genomic DNA was purified and abasic sites were quantified using an aldehyde-reactive probe. Results are an average of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Knockdown, Infection, Expressing, shRNA, Sequencing, Single Cell Gel Electrophoresis, Stable Transfection, Plasmid Preparation, Purification

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Ectopic Expression of BCL11A 160–520 Accelerates DNA Repair and Increases Resistance to H 2 O 2 . ( A ) RPE1 cells expressing the BCL11A 160–520 peptide were exposed to 100 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( B ) RPE1 cells expressing the BCL11A 160–520 peptide were treated with 0, 50 or 100 μM H 2 O 2 and submitted to a clonogenic assay. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Expressing, Single Cell Gel Electrophoresis, Clonogenic Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A cooperates with RAS to transform primary cells and escape senescence. IMR90 primary fibroblastic cells were infected with retroviruses expressing either HRAS alone or HRAS and BCL11A 160–520 , as indicated. ( A ) Total cell extracts were analyzed by immunoblotting with the indicated antibodies. ( B ) Cells were plated in soft agar 2 days after infection and colonies were counted after 2 weeks Results are an average of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) IMR90 cells were selected for 5 days with puromycin, cells were then submitted to single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( D ) β-Gal associated senescence was measured 7 days after infection with vectors expressing HRAS or HRAS and BCL11A 160–520 . Values were normalized to the value of empty vector. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( E ) Expression of the senescence markers P16, P21, IL6 and IL8 was measured by RT-PCR 5 days following infection of IMR90 cells with vectors expressing either HRAS, BCL11A 160–520 , HRAS and BCL11A 160–520 or nothing. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: Other antibodies used are BCL11A 382A (1:4000, Bethyl), NTHL1 (1:1000, Proteintech), anti-GST (1:1000, Abcam), anti-His (1:3000, Sigma), anti-HA (1:1000 Covance) and anti-γ-tubulin (1:10 000, Sigma).

Techniques: Infection, Expressing, Western Blot, Single Cell Gel Electrophoresis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction