Journal: Frontiers in Oncology

Article Title: Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells

doi: 10.3389/fonc.2020.01666

Figure Lengend Snippet: Genomic alterations based on whole exome sequencing data. (A) Copy number loss of HMGN2 in Het-1A-Smoke-8M cells. (B) An IGV snapshot of MED1 gene representing non-synonymous SNV (pL666F). (C) Proliferation curve of Het-1A-Smoke-8M cells with and without exogenous overexpression of HMGN2 and MED1 (*** p < 0.0001, ** p < 0.01, ns, statistically not significant). (D) Invasive potential of Het-1A-Smoke-8M cells with and without exogenous overexpression of HMGN2 and MED1 (** p < 0.01).

Article Snippet: Monoclonal rabbit anti-HMGN2 antibody (Cat#[EPR7091] ab124997, Abcam) was used as a primary antibody at a dilution of 1:200.

Techniques: Sequencing, Over Expression

Journal: Frontiers in Oncology

Article Title: Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells

doi: 10.3389/fonc.2020.01666

Figure Lengend Snippet: Immunohistochemical validation of HMGN2 in ESCC tissue sections and adjacent normal. Tissue sections from ESCC and adjacent normal were stained with anti-HMGN2 antibody. Scale bar represents 100 μm.

Article Snippet: Monoclonal rabbit anti-HMGN2 antibody (Cat#[EPR7091] ab124997, Abcam) was used as a primary antibody at a dilution of 1:200.

Techniques: Immunohistochemical staining, Biomarker Discovery, Staining

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: PHA induced HMGN2 expression in PBMCs. PBMCs were seeded at a density of 1 × 10 7 per well in 6 well plates and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Normal medium was used as the control. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and stained with anti-CD3-PE and anti-HMGN2-IgG-FITC, followed to run on a Beckman coulter FC500 Flow cytometry. Data were analyzed by using Submit 5.2 software after gate lymphocytes (R5) group on dot plot graph. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD3 + T cells. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to medium control (p < 0.05). (D) Representative plots of the isotype staining and 2 nd -Ab-FITC staining control.

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Software

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: PHA induced HMGN2 expression in different cell populations. PBMCs were seeded at a density of 1 × 10 7 per well in 6 well plates and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Normal medium was used as the control. Cells were collected and stained (A) Anti-CD4-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained; (B) Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. Figures are representative of three independent experiments. (C, D) Error bars represent HMGN2 intracellular expression positive rate (%) in CD4 + or CD8 + T cell populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to IL-2 and medium control (p < 0.05), #Significantly higher compared to CD4 + T cell population (p < 0.05). (E) Representative plots of the isotype staining and 2 nd -Ab-FITC staining control.

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Expressing, Control, Staining

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: PHA-induced HMGN2 was mainly expressed in CD8 + T cell population. PBMCs were seeded at a density of 1 × 10 7 per well in 6 well plates and stimulated with 20 μg/ml PHA for 72 hours. Cells were collected and stained with CD3-FITC and CD8-PE. CD3 + CD8 + T cells (Gate R1), CD3 + CD8 - T cells (Gate R2) and CD3 - mixed cells (Gate R3) were purified with MoFlo XDP sorter. 4 × 10 6 CD3 + CD8 + T cells, CD3 + CD8 - T cells and CD3 - mixed cells were incubated in 24-well plates respectively for 5 days. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD8 + T cells populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to CD8 - CD3 + and MIX cells (p < 0.05).

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Staining, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: HMGN2 expression in Tumor antigen stimulated PBMCs. PBMCs were seeded at a density of 1 × 10 7 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. Normal medium was used as the control. (A) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (B) Error bars represent HMGN2 intracellular expression positive rate (%) in PBMCs after stimulation with T-Ag. (C) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. Data are represented as means ± SD of three independent experiments.

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Expressing, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: T-Ag activated T cells and induced HMGN2 expression in activated T cell populations. PBMCs were seeded at a density of 1 × 10 7 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. Normal medium was used as the control. Cells were removed and (A) stained with CD44-APC/CD8-PE/intracellular HMGN2-2 nd -Ab-FITC. (Aa) Gate 1 (CD44 high ) as the activated T cells, gate R6 (CD44 low ) as the naïve T cells. (Ab-Ac) Intracellularly expression of HMGN2 in CD44 high activated CD8 + T cells (Ab) and CD44 low naïve CD8 + T cells (Ac) . (B) Error bars represented the percentage of activation PBMCs after stimulated with T-Ag. (C) Error bars represented HMGN2 intracellular expression positive rate (%) in T-Ag-activated CD8 + T cell populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to medium control (p < 0.05). #Significantly higher compared to CD44 low CD8 + naïve T cells (p < 0.05).

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Expressing, Control, Staining, Activation Assay

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: T-Ag activated CD8 + T cells released HMGN2 to kill tumor cells. PBMCs were seeded at a density of 1 × 10 7 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. (A) Cells were removed and stained with CD44-APC/CD8-FITC. CD44 high /CD8 + T activated T cells (Gate R4) were purified with MoFlo XDP sorter. The purified CD44 high /CD8 + T cells were cultured in complete medium with 2000 IU/ml IL-2 for 5 days. (B) The cells were removed and stained with Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. (C) The antitumor effects of the supernatants at different concentration. (D) The antitumor effects of the 20% (v/v) supernatants after blocking HMHN2 using anti-HMGN2 antibody. Figures are representative of three independent experiments. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to medium control (p < 0.05). #Significantly decreased after with anti-HMGN2 antibody (p < 0.05).

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Staining, Purification, Cell Culture, Concentration Assay, Blocking Assay, Control

Journal: Molecular Cancer

Article Title: HMGN2, a new anti-tumor effector molecule of CD8 + T cells

doi: 10.1186/1476-4598-13-178

Figure Lengend Snippet: HMGN2, released by T-Ag activated CD8 + T cells, transmembrane transported into tumor cells. HMGN2 protein and the supernatant of T-Ag activated CD8 + T cells were pre-labeled with FITC. Tca8113 cells were seeded at a density of 3 × 10 4 per well in 24-well plates. After overnight growth, the cells were cultured in medium with FITC pre-labeled samples. (A) HMGN2 transport into tumor cells analyzed with fluorescence microscope. The three figures are the same area. (a) Light micrographs of Tca8113 cells. (b) Fluorescent micrographs of Tca8113 cells of Hoechst 33258 nuclear staining. (c) Fluorescent micrographs of FITC labeled HMGN2 protein distribution in Tca8113 cells. (B) The Tca8113 cells were analyzed with fluorescent microscope. (a, b, c) FITC pre-labeled HMGN2 as the positive control. (d, e, f) FITC pre-labeled CD8 + T cells supernatant. (a, d) Cells under a light microscope. (b, e) Cells under a fluorescent microscope. (c, f) Cells under a fluorescent microscope after cultured in medium with HMGN2 depleted samples. (C) The Tca8113 cells were analyzed with Flow Cytometry. (a) Untreated Tca8113 control. (b, d) Tca8113 cultured in medium with FITC labeled samples. (c, e) Tca8113 cells cultured in medium with HMGN2 depleted samples. Figures are representative of three independent experiments. (f) Error bars represent FITC positive rate (%) of Tca8113 cells after cultured in medium with FITC labeled or HMGN2 depleted sample for 1 hour. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to HMGN2 undepleted (p < 0.05).

Article Snippet: After washing three times with wash buffer, 100 μl of rabbit anti-human HMGN2 antibody (Proteintech Group, USA) (1:500) were added and plates were incubated at 37°C for 1 hour.

Techniques: Labeling, Cell Culture, Fluorescence, Microscopy, Staining, Positive Control, Light Microscopy, Flow Cytometry, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: Validation of potential novel Aurora B phosphorylation sites and substrate. A, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B (wild-type and kinase-dead), in the presence of ATPγS. Recombinant GST-INCENP 1–80, GST-INCENP 1–80S72A, or histone H3 was added as substrate. B, U2OS cells were transfected with either empty GFP vector (ev) or a plasmid encoding GFP-HMGN2. Mitotic cells were collected by mitotic shake-off, and the remaining adherent cells were used as interphase cell input. The overexpressed proteins were immunoprecipitated from the interphase or mitotic cell lysates with an anti-GFP antibody. The precipitated proteins were separated by SDS-PAGE, and Western blots were probed with an antibody specific for phosphorylated Ser-25/Ser-29 in HMGN2 (middle panel) and subsequently reprobed with an anti-GFP antibody (upper panel). Western blots of whole cell extracts were probed with an antibody specific for phosphorylated histone H3 (Ser-10, lower panel). C and D, U2OS cells were released into nocodazole in the presence or absence of the indicated Aurora B inhibitors. HMGN2, Aurora B, and centromeres (CREST) were visualized with specific antibodies. Images of fields of mitotic cells were captured by a Zeiss LSM microscope (C), and the fraction of mitotic cells with no, low, or high levels of HMGN2 on mitotic chromosomes was quantified (D). For each condition >100 cells were counted. Scale bar = 20 micrometer.

Article Snippet: For HMGN2 immunofluorescence, a brief PEM/Triton 0.2% pre-extraction was performed prior to fixation with 4% PFA, and rabbit anti-HMGN2 polyclonal antibody (Acris Antibodies) was used as primary antibody.

Techniques: In Vitro, Kinase Assay, Recombinant, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Microscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: Aurora B substrates unique to this screen The criteria for inclusion include phosphopeptide(s) found in two independent experiments, R X (S/T) motif, E-score ≤ 10E-4, SLIP score ≥ 6, and substrate not found by Kettenbach et al. ( 30 ).

Article Snippet: For HMGN2 immunofluorescence, a brief PEM/Triton 0.2% pre-extraction was performed prior to fixation with 4% PFA, and rabbit anti-HMGN2 polyclonal antibody (Acris Antibodies) was used as primary antibody.

Techniques: Binding Assay

Journal: Molecular & Cellular Proteomics

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex

doi: 10.1074/mcp.m111.013912

Figure Lengend Snippet: FIG. 6. Validation of potential novel Aurora B phosphorylation sites and substrate. A, in vitro kinase assay with recombinant His- INCENP/GST-tagged Aurora B (wild-type and kinase-dead), in the presence of ATPS. Recombinant GST-INCENP 1–80, GST-INCENP 1–80S72A, or histone H3 was added as substrate. B, U2OS cells were transfected with either empty GFP vector (ev) or a plasmid encoding GFP-HMGN2. Mitotic cells were collected by mitotic shake-off, and the remaining adherent cells were used as interphase cell input. The overexpressed proteins were immunoprecipitated from the interphase or mitotic cell lysates with an anti-GFP antibody. The precipitated proteins were separated by SDS-PAGE, and Western blots were probed with an antibody specific for phosphorylated Ser-25/Ser-29 in HMGN2 (middle panel) and subsequently reprobed with an anti-GFP antibody (upper panel). Western blots of whole cell extracts were probed with an antibody specific for phosphorylated histone H3 (Ser-10, lower panel). C and D, U2OS cells were released into nocodazole in the presence or absence of the indicated Aurora B inhibitors. HMGN2, Aurora B, and centromeres (CREST) were visualized with specific antibodies. Images of fields of mitotic cells were captured by a Zeiss LSM microscope (C), and the fraction of mitotic cells with no, low, or high levels of HMGN2 on mitotic chromosomes was quantified (D). For each condition 100 cells were counted. Scale bar 20 micrometer.

Article Snippet: 48 Molecular & Cellular Proteomics 11.5 Membranes were blocked with 4% milk in Tris-buffered saline containing 1% Tween 20 (TBST) prior to incubation with the following primary antibodies in 4% milk-TBST: mouse anti-GST (GE Healthcare), rabbit anti-Ser(P)-10 histone H3 (Upstate Biotechnology, Charlottesville, VA), mouse anti-FLAG (Campro Scientific, Berlin, Germany), rabbit anti-GFP (gift of The Netherlands Cancer Institute), mouse anti-Aurora B (BD Biosciences, Breda, The Netherlands), mouse anti-vesicular stomatitis virus (Sigma-Aldrich), rabbit anti-thiophosphate ester (Epitomics), or anti-Ser(P)-25/29 HMGN2 (Acris Antibodies, Herford, Germany).

Techniques: Biomarker Discovery, Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Microscopy

Journal: bioRxiv

Article Title: Increasing cell size remodels the proteome and promotes senescence

doi: 10.1101/2021.07.29.454227

Figure Lengend Snippet: (a) Distribution of slopes derived from HLF cells for proteins associated with the indicated compartments. Violin plots depict the average slopes for the proteins highlighted in . P-values above the violin plots are derived from a t-test between the indicated protein group and the rest of the dataset. t-tests comparing the slopes for each group of proteins are visualized in a grid format. (b) Replicate experiment using the immortalized hTERT RPE-1 cell line was performed as in (a). (c) Validation of lysosome super-scaling with cell size using flow cytometry. Both the lysosomal protein LAMP1 and the Lysotracker dye amount increase with cell size faster than Actin, which is a proxy for total protein. The data for G1 hTERT RPE-1 cells were binned by cell size (SSC, the side scatter parameter) and plotted as mean protein amounts per cell for each size bin (solid lines). Dark shaded area shows standard error of the mean for each bin, and light shaded area shows the standard deviation. A representative is shown of n=5 biological replicates for each experiment. About 100,000 cells were analyzed for each sample. (d) Examples from our proteomics data set of cell-size-dependent protein concentration changes in proliferating cells that are normally associated with senescence. (e) RB is diluted with increasing G1 cell size.

Article Snippet: We used the following primary antibodies: HMGB1 (Abcam, ab79823), HMGN2 (CST, 9437S), RPLP0 (Sigma, SAB1402899), Actin (Sigma-Aldrich, A2103), UCHL1 (CST, 13179S), VAT1 (Santa Cruz, sc-515705), LAMP1 (CST, 9091), alpha-Tubulin (Abcam, ab6160).

Techniques: Derivative Assay, Biomarker Discovery, Flow Cytometry, Standard Deviation, Protein Concentration