Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module.

doi: 10.1016/j.bbagrm.2023.194929

Figure Lengend Snippet: Fig. 4. SAGA's HAT module regulates SAGA DUB activity independent of enzymatic activity. A: Quantitation of western blots of SAGA DUB activity on recombinant H2B-Ub nucleosomes (400 nM) under conditions of limiting SAGA enzyme (25 nM) with and without the SWIRM domain. B: Quantitation of western blots of SAGA DUB activity on HeLa nucleosomes with ~50 nM H2B ubiquitinated nucleosome. SAGA DUB activity is compared with and without the SWIRM domain, with excess SAGA, 100 nM. C: WT SAGA DUB (25 nM) activity on recombinant H2B-Ub nucleosomes (400 nM) in absence and presence of acetyl CoA (10 μM). D: Quantitation of C and replicate.

Article Snippet: Experiments done with excess ubiquitinated nucleosome used purified recombinant mononucleosomes containing histone H2B ubiquitinated at K120 (H2B-K120Ub) (EpiCypher, 16-0370) at 400 nM and 25 nM SAGA.

Techniques: Activity Assay, Quantitation Assay, Western Blot, Recombinant

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) Schematic representation of the MTB MIC Stat3 –/– Chi3l1 OE GEMM used in this study. Created in BioRender (Muller W, 2026, https://BioRender.com/avc83im ). ( B ) Representative hematoxylin and eosin (H&E) staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction. ( C ) Quantification of hyperplastic area in WT ( n = 12), Chi3l1 OE ( n = 5), Stat3 –/– ( n = 11), and Stat3 –/– Chi3l1 OE ( n = 12) MIC mammary glands at 2 weeks after induction. Represented as percentage of total mammary gland area. ( D ) Quantification of hyperplastic area in WT ( n = 11), Chi3l1 OE ( n = 7), Stat3 –/– ( n = 11), and Stat3 –/– Chi3l1 OE ( n = 11) MIC mammary glands at 6 weeks after induction. Represented as percentage of total mammary gland area. ( E ) Representative H&E staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 6 weeks after induction. ( F ) Mammary tumor onset in WT ( n = 29), Chi3l1 OE ( n = 20), Stat3 –/– ( n = 40), and Stat3 –/– Chi3l1 OE ( n = 28) MIC mice, tracked by weekly physical palpation. Analysis by log-rank test. ( G ) Summary of mammary tumor onset and penetrance in WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mice. * P < 0.05, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test. Scale bars: 500 μm.

Article Snippet: Immunoblot was performed as previously described ( , ) using the following antibodies: mouse CHI3L1 (1:1,000; Invitrogen PA5-8135), STAT3 (1:1,000; Cell Signaling 9139), p-STAT3 (1:1,000; Cell Signaling 9145), CitH3 (1:1,000; Abcam Ab5103), vinculin (1:2,000; Millipore MAB3574), and α-tubulin (1:1,000; Cell Signaling 3873).

Techniques: Staining

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) Staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction for CD8, granzyme B (GZMB), CD3, Pan-CK, and DAPI. ( B ) RNA FISH against IFN-γ with staining for CD3, Pan-CK, and DAPI on mammary tissue from WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction. ( C – G ) Quantification of total CD3 + CD8 + , CD3 + CD8 + GZMB + , and tumor-infiltrating CD3 + CD8 + T cells, CD3 + IFN-γ + cells, and Ly6G + cells in WT ( n = 5), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 6), and Stat3 –/– Chi3l1 OE ( n = 9) MIC mammary glands at 2 weeks after induction. ( H ) Staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction using antibodies against Ly6G, MPO, NE, pan-CK, and DAPI. ( I and J ) Quantification of total Ly6G + MPO + cells and Ly6G + MPO + NE + cells in WT ( n = 5), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 6), and Stat3 –/– Chi3l1 OE ( n = 9) MIC mammary glands at 2 weeks after induction. ( K ) Quantification of CitH3 immunoblots normalized to α-tubulin. ( L ) Immunoblots for CitH3 and α-tubulin on WT ( n = 3), Chi3l1 OE ( n = 3), Stat3 –/– ( n = 3), and Stat3 –/– Chi3l1 OE ( n = 3) MIC mammary glands at 2 weeks after induction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test. Scale bars: 100 μm.

Article Snippet: Immunoblot was performed as previously described ( , ) using the following antibodies: mouse CHI3L1 (1:1,000; Invitrogen PA5-8135), STAT3 (1:1,000; Cell Signaling 9139), p-STAT3 (1:1,000; Cell Signaling 9145), CitH3 (1:1,000; Abcam Ab5103), vinculin (1:2,000; Millipore MAB3574), and α-tubulin (1:1,000; Cell Signaling 3873).

Techniques: Staining, Western Blot

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) H&E staining of mammary glands from IgG2a- and anti-Ly6G–treated MIC Stat3 –/– Chi3l1 OE mice at 2 weeks after induction. ( B ) Quantification of hyperplastic area in IgG2a-treated ( n = 8) and anti-Ly6G–treated ( n = 8) MIC Stat3 –/– Chi3l1 OE mice. Represented as percentage of mammary gland area. ( C ) Staining of mammary tissue from MIC Stat3 –/– Chi3l1 OE mice treated with anti-Ly6G or IgG2a for 2 weeks, for GZMB, CD8, CD3, pan-CK, and DAPI. ( D – F ) Quantification of CD3 + CD8 + , CD3 + CD8 + GZMB + , and tumor-infiltrating CD3 + CD8 + T cells in IgG2a-treated ( n = 8) and anti-Ly6G–treated ( n = 8) MIC Stat3 –/– Chi3l1 OE mammary glands. ( G ) Staining of mammary tissue from MIC Stat3 –/– Chi3l1 OE mice treated with anti-Ly6G or IgG2a, for PD-1, CD4, CD3, pan-CK, and DAPI. ( H – J ) Quantification of CD3 + CD4 + , CD3 + CD4 + PD-1 + , and tumor-infiltrating CD3 + CD4 + T cells in IgG2a-treated ( n = 8) and anti-Ly6G–treated ( n = 8) MIC Stat3 –/– Chi3l1 OE mammary glands. ( K ) H&E staining of mammary glands from IgG2a- and anti-Ly6G–treated MIC Chi3l1 OE mice at 2 weeks after induction. ( L ) Quantification of hyperplastic area in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mice. Represented as percentage of mammary gland area. ( M ) Staining of mammary tissue from MIC Chi3l1 OE mice treated with anti-Ly6G or IgG2a for 2 weeks, for GZMB, CD8, CD3, pan-CK, and DAPI. ( N – P ) Quantification of CD3 + CD8 + , CD3 + CD8 + GZMB + , and tumor-infiltrating CD3 + CD8 + T cells in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mammary glands. ( Q ) Staining of mammary tissue from MIC Chi3l1 OE mice treated with anti-Ly6G or IgG2a for 2 weeks, for PD-1, CD4, CD3, pan-CK, and DAPI. ( R – T ) Quantification of CD3 + CD4 + , CD3 + CD4 + PD1 + , and tumor-infiltrating CD3 + CD4 + T cells in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mammary glands. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by unpaired Student’s t test. Scale bars: 100 μm in A , C , G , M , and Q ; 500 μm in K .

Article Snippet: Immunoblot was performed as previously described ( , ) using the following antibodies: mouse CHI3L1 (1:1,000; Invitrogen PA5-8135), STAT3 (1:1,000; Cell Signaling 9139), p-STAT3 (1:1,000; Cell Signaling 9145), CitH3 (1:1,000; Abcam Ab5103), vinculin (1:2,000; Millipore MAB3574), and α-tubulin (1:1,000; Cell Signaling 3873).

Techniques: Staining

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) H&E staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC lungs at mammary tumor endpoint. ( B ) Percentage of WT ( n = 12), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 13), and Stat3 –/– Chi3l1 OE ( n = 11) MIC mice with pulmonary metastases. ( C ) Quantification of metastatic area (percent total lung area) in WT ( n = 12), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 13), and Stat3 –/– Chi3l1 OE ( n = 11) MIC lungs. ( D ) Quantification of PyMT + cells (percent live cells in blood) of WT ( n = 4) and Chi3l1 OE ( n = 3) MIC mice. ( E ) FACS for circulating PyMT + cells in WT and Chi3l1 OE MIC blood at 2 weeks after induction. ( F ) Staining of WT and Chi3l1 OE MIC mammary glands at 2 weeks after induction for laminin, collagen IV, pan-CK, and DAPI. ( G and H ) Quantification of area occupied by laminin or collagen IV (percent total mammary gland area) in WT ( n = 7) and Chi3l1 OE ( n = 8) MIC mammary glands. ( I ) FACS for circulating PyMT + cells in blood of Chi3l1 OE MIC mice after anti-Ly6G or IgG2a treatment for 2 weeks. ( J ) Quantification of PyMT + cells (percent live cells in the blood) of Chi3l1 OE MIC mice treated with IgG2a ( n = 6) or anti-Ly6G ( n = 5). ( K ) Staining of mammary tissue from MIC Chi3l1 OE mice treated with anti-Ly6G or IgG2a for laminin, collagen IV, pan-CK, and DAPI. ( L and M ) Quantification of area occupied by laminin and collagen IV (percent mammary gland area) in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mammary glands. ( N ) Schematic of Transwell invasion assay through synthetic ECM. Created in BioRender (Muller W, 2026, https://BioRender.com/4lhz2a3 ). ( O ) ECM-invading PyMT + cells with PBS, rmChi3l1, or murine neutrophils, stained with crystal violet. Neutrophils + PBS (no PyMT cells) is shown as negative control. ( P ) Quantification of invading PyMT + cells. n = 3 technical replicates for each condition. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test ( C and P ) or by unpaired Student’s t test (for D , G , H , J , L , and M ). Scale bars: 1 cm in A , left panels; 1 mm in A , right panels; 100 μm in F , K , and O .

Article Snippet: Immunoblot was performed as previously described ( , ) using the following antibodies: mouse CHI3L1 (1:1,000; Invitrogen PA5-8135), STAT3 (1:1,000; Cell Signaling 9139), p-STAT3 (1:1,000; Cell Signaling 9145), CitH3 (1:1,000; Abcam Ab5103), vinculin (1:2,000; Millipore MAB3574), and α-tubulin (1:1,000; Cell Signaling 3873).

Techniques: Staining, Transwell Invasion Assay, Negative Control

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) Immunoblots for Chi3l1, Stat3, and α-tubulin on WT ( n = 7) and Chi3l1 –/– ( n = 6) MIC mammary endpoint tumors. ( B ) Representative H&E staining of WT and Chi3l1 –/– MIC lungs at mammary tumor endpoint. ( C ) Percentage of WT ( n = 11) and Chi3l1 –/– ( n = 11) MIC mice with pulmonary metastases. ( D ) Quantification of total pulmonary metastatic area in WT ( n = 11) and Chi3l1 –/– ( n = 11) MIC lungs. Represented as percentage of total lung area. ( E ) Number of lung metastatic lesions in WT ( n = 11) and Chi3l1 –/– ( n = 11) MIC lungs. ( F ) Staining of WT and Chi3l1 –/– MIC mammary glands at 2 weeks after induction, for Ly6G, MPO, NE, pan-CK, and DAPI. ( G and H ) Quantification of Ly6G + cells and Ly6G + MPO + NE + cells in WT ( n = 7) and Chi3l1 –/– ( n = 10) MIC mammary glands at 2 weeks after induction. ( I ) Staining of WT and Chi3l1 –/– MIC mammary glands at 2 weeks after induction, for laminin, collagen IV, pan-CK, and DAPI. ( J and K ) Quantification of area occupied by laminin or collagen IV in WT ( n = 7) and Chi3l1 –/– ( n = 10) MIC mammary glands at 2 weeks after induction. Represented as percentage of total mammary gland area. ( L ) FACS sorting for PyMT + tumor cells in the blood of WT MIC mice treated with anti-Chi3l1 neutralizing antibody or IgG2b isotype control at 2 weeks after induction. ( M ) Quantification of PyMT + cells as percentage of total live cells in the blood of WT MIC mice treated with IgG2b ( n = 5) or anti-Chi3l1 ( n = 5) for 2 weeks. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by unpaired Student’s t test. Scale bars: 1 mm in B , left panels; 100 μm in B , right panels; 10 μm in F and I .

Article Snippet: Immunoblot was performed as previously described ( , ) using the following antibodies: mouse CHI3L1 (1:1,000; Invitrogen PA5-8135), STAT3 (1:1,000; Cell Signaling 9139), p-STAT3 (1:1,000; Cell Signaling 9145), CitH3 (1:1,000; Abcam Ab5103), vinculin (1:2,000; Millipore MAB3574), and α-tubulin (1:1,000; Cell Signaling 3873).

Techniques: Western Blot, Staining, Control

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A and B ) Staining of human ER + or HER2 + breast cancer samples for CHI3L1, p-STAT3, pan-CK, and DAPI. Micrograph represents areas of high CHI3L1 and low CHI3L1 from different patient samples. ( C and D ) Pearson’s correlation between levels of epithelial CHI3L1 and epithelial p-STAT3 in human ER + ( n = 89) and HER2 + ( n = 85) breast cancer patient samples. ( E ) IHC staining of human primary breast tumor and matched metastatic samples, for CHI3L1, counterstained with hematoxylin. Side-by-side pictures are derived from the same patient. ( F ) Quantification of CHI3L1 + cells in patient-matched primary breast tumors ( n = 10) and recurrent metastatic tumors ( n = 10). ( G ) Summary of the proposed model for Chi3l1-mediated cancer cell dissemination. Cancer cells secrete Chi3l1 that recruits neutrophils, which degrade the ECM and enhance CTCs. Created in BioRender (Muller W, 2026, https://BioRender.com/kk5b3ud ). * P < 0.05, paired Student’s t test. Scale bars: 100 μm.

Article Snippet: Immunoblot was performed as previously described ( , ) using the following antibodies: mouse CHI3L1 (1:1,000; Invitrogen PA5-8135), STAT3 (1:1,000; Cell Signaling 9139), p-STAT3 (1:1,000; Cell Signaling 9145), CitH3 (1:1,000; Abcam Ab5103), vinculin (1:2,000; Millipore MAB3574), and α-tubulin (1:1,000; Cell Signaling 3873).

Techniques: Staining, Immunohistochemistry, Derivative Assay

Journal: BMC Molecular Biology

Article Title: RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX

doi: 10.1186/1471-2199-10-55

Figure Lengend Snippet: RNF168 resides in detergent-insoluble structures and interacts with the histone core . A) Before fixing, HeLa cells transfected with the GFP-tagged indicated constructs or the vector alone, were pre-treated (right panels) or not (left panels) with Triton X-100. The nucleus was stained with To-Pro 3. B) To test RNF168 capability to associate with histones, we performed in vitro pull down assay on cell lysate derived from 293T cells expressing the GFP-tagged forms of histones H2A, H2B, H3 and H4. To detect histone binding, the associated proteins were resolved by SDS-PAGE and analysed by anti-GFP immunoblot. To normalize for equal loading, Ponceau-red staining is shown.

Article Snippet: Antibodies used were: mouse monoclonal anti-FLAG and anti-FLAG affinity gel (M2, Sigma), mouse monoclonal anti RNF168 (Abcam), rabbit Phospho-(Ser/Thr) ATM/ATR Substrate Antibody (Cell Signaling), mouse monoclonal anti-Ub P4D1 (Santa Cruz) and FK2 (Stressgen Bioreagents), mouse monoclonal anti-ubiquityl-Histone H2A (Upstate), mouse monoclonal anti-ubiquityl-Histone H2B (Upstate), mouse monoclonal anti-GFP (Santa Cruz), rabbit polyclonal anti-GST was home made, anti phospho-Histone H2A.X (Ser139; Upstate).

Techniques: Transfection, Construct, Plasmid Preparation, Staining, In Vitro, Pull Down Assay, Derivative Assay, Expressing, Binding Assay, SDS Page, Western Blot

Journal: BMC Molecular Biology

Article Title: RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX

doi: 10.1186/1471-2199-10-55

Figure Lengend Snippet: RNF168 ubiquitinates histone H2A but not H2B, both in vitro and in vivo . A) The in vitro ubiquitination assay was performed with GST-RNF168 wild type and the RF* mutant, using recombinant histones H2A and H2B as substrates. Refer to Methods for details. The reaction mixtures were solved by SDS-PAGE and the immunoblot with antibodies uH2A and uH2B shows the monoubiquitinated forms of histones H2A and H2B, respectively. TCL (Total Cell Lysates) are loaded to validate the signals. Immunoblots directed to RNF168 antibodies were done as control of equal loading. B) 293T cells were co-transfected with GFP alone, GFP-RNF168 WT or RF* together with FLAG-tagged histones H2A (left panels) or H2B (right panels). A small amount (1/20) of the transfected cells were lysed with standard procedure to verify protein expression level (IB using GFP and FLAG antibodies, lower panels). The remaining part was subjected to acid extraction, and the histone component was analysed by SDS-PAGE and immunodecorated as indicated. (◆) indicates the mono-ubiquitinated form of histones H2A and H2B; (◆) di- and (◆◆◆) tri-ubiquitinated forms are visible when the wild type RNF168 is expressed, but not in the presence of the RF* mutant or the vector alone. No signal of di- and tri-ubiquitination was detected for H2B. C) Cells transfected with the indicated GFP constructs were immunostained with anti-uH2A. D) 293T cells were co-transfected with cDNA encoding the GFP-RNF168 (WT) or the vector alone and the FLAG-H2A, and subjected to acid extraction. Proteins were resolved by SDS-PAGE and immunoblotted with FK2 (left panel) and Apu3.A8 (K63-chain specific, right panel) anti-Ub antibodies.

Article Snippet: Antibodies used were: mouse monoclonal anti-FLAG and anti-FLAG affinity gel (M2, Sigma), mouse monoclonal anti RNF168 (Abcam), rabbit Phospho-(Ser/Thr) ATM/ATR Substrate Antibody (Cell Signaling), mouse monoclonal anti-Ub P4D1 (Santa Cruz) and FK2 (Stressgen Bioreagents), mouse monoclonal anti-ubiquityl-Histone H2A (Upstate), mouse monoclonal anti-ubiquityl-Histone H2B (Upstate), mouse monoclonal anti-GFP (Santa Cruz), rabbit polyclonal anti-GST was home made, anti phospho-Histone H2A.X (Ser139; Upstate).

Techniques: In Vitro, In Vivo, Ubiquitin Proteomics, Mutagenesis, Recombinant, SDS Page, Western Blot, Control, Transfection, Expressing, Extraction, Plasmid Preparation, Construct