Journal: Journal of Cancer

Article Title: The Quantum of Initial Transformed Cells Potentially Modulates the Type of Local Inflammation Mechanism Elicited by Surrounding Normal Epithelial Tissues and Systemic Immune Pattern for Tumor Arrest or Progression

doi: 10.7150/jca.10787

Figure Lengend Snippet: Angiogenesis, inflammation and expression pattern of redox forms of HMGB1 in NTAT : (A) To be certain that harvested NTAT were not contaminated with tumor tissues, random sections of harvested NTAT from each group were H & E stained and critically examined under the microscope (Olympus BX41, Japan). Corresponding tumor samples from animals that developed tumors were used for comparison. All NTAT were free from tumor cells/tissue contamination. Endothelial cell proliferation/ angiogenesis (arrows) was common in the NTAT of tumor bearing mice but not in non-tumor or control mice. (B) VEGF-A protein expression levels in NTAT of tumor (T) and non-tumor (nT) bearing mice. Expression levels were quantified by densitometric measurements using ImageJ software. Bars represent mean ± SEM. (C) IL-6 expression in NTAT of tumor (T) bearing and non-tumor (nT) bearing. (D) Visual time course observation of tissue inflammation, tumor arrest or progression in two groups of mice ( n = 12 per group) that were either inoculated with 1 × 10 3 cancer cells (MT) or 5 × 10 3 cancer cells (Above-MT) and sacrificed at the indicated time points. To aid visualization of inflamed areas and implanted cancer cells, NTAT from MT mice were fixed in 4% paraformaldehyde for 3 days and then visualized on a transluminator (Jim-X Scientific, China). Arrows indicate implanted cells. (E) Time course expression levels of (i) total HMGB1 and (ii) redox forms of HMGB1 in the NTAT of "MT" and "Above-MT" groups. For all Western blot panels, detection of actin served as the loading control. Immunoblots and macrographs are representative of three independent experiments.

Article Snippet: Primary antibodies used were rabbit polyclonal anti -HMGB1, -IL-1β, -IL-6, -IFN-γ (Proteintech, USA), and rabbit anti-VEGF-A (Bioworld, USA).

Techniques: Expressing, Staining, Microscopy, Comparison, Control, Software, Western Blot

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: A mild heat shock (HS) pretreatment attenuates oxidative stress (H2O2)-induced HMGB1 release in macrophages cultures. A, Effects of mild or severe heat shock on HMGB1 release. RAW 264.7 cells were subjected to a brief heat shock (1 h) at 42.5°C (mild heat shock), 43.5°C or higher (severe heat shock), after which was returned to 37°C and incubated for additional 12 h. HMGB1 in the culture medium were detected by Western blotting analysis (top). In parallel, the cell viability was determined by LDH release assay (bottom). Ctrl, control cells. Blot is representative of three experiments with similar results. Values are mean ± SEM (n = 3) of three experiments in duplicate. B, Mild HS pretreatment induced Hsp expression. RAW 264.7 cells were subjected to heat shock (42.5°C, 1 h), and cellular levels of Hsp90 and Hsp72 were detected by Western blotting analysis at the indicated time points after heat shock. In parallel, levels of HMGB1 in the culture medium were determined by Western blotting analysis. Tubulin was used as a loading control. Values are representative of three independent experiments with similar results. C, Effects of oxidative stress on Hsp72 expression. RAW 264.7 cells were stimulated with H2O2 at nontoxic (0.125 mM), or low-toxic (0.25 mM) concentrations, and cellular Hsp72 were detected at 12 h poststimulation by Western blotting analysis. GAPDH was used as a loading control. Values are representative of three independent experiments with similar results. D, Mild heat shock pretreatment inhibited H2O2-induced HMGB1 release. After mild heat shock (42.5°C, 1 h), cells were allowed to recover for 12 h at 37°C and then stimulated for 12 h with H2O2 at nontoxic (0.125 mM) or low-toxic (0.25 mM) concentrations. Levels of HMGB1 in the culture medium were determined by Western blotting and expressed (in arbitrary units; AU) as mean ± SEM of three experiments in duplicate. In parallel, the cell viability was determined by MTT assay, and expressed as mean ± SEM (n = 4) of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Incubation, Western Blot, Lactate Dehydrogenase Assay, Control, Expressing, MTT Assay

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Overexpression of Hsp72 renders macrophages resistant to H2O2-induced HMGB1 release. A, Western blotting analysis of Hsp72 levels in RAW 264.7 cells transfected with empty plasmid (lane 1), Hsp72 expression construct (lane 2) or challenged with mild heat shock (HS, 42.5°C, 1 h). Tubulin was used as a loading control. B, Visualization of fluorescent Hsp72 protein in RAW 264.7 cells transfected with Hsp72 expression construct (top), or empty plasmid (bottom). Nuclei were visualized by Hoechst staining. C, Western blotting analysis of H2O2-induced HMGB1 release in RAW 264.7 cells transfected with empty plasmid, or Hsp72 expression construct. RAW 264.7 cells were stimulated with H2O2 (0.125 and 0.25 mM) for 12 h, and HMGB1 levels in the culture medium were determined, and expressed as mean ± SEM of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Construct, Control, Staining

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Overexpression of Hsp72 attenuates H2O2-induced HMGB1 cytoplasmic translocation in macrophage cultures. A and B, H2O2 induces transient nuclear translocation of Hsp72 in macrophages cultures. pcDNA3.1-Hsp72-transfected RAW 264.7 cells were stimulated with H2O2 (0.125 mM) for various period of time and examined for Hsp72 subcellular localization by immunocytochemistry (A) or cell fractionation/Western blot (B). Green, Hsp72; blue, nuclei. Original magnification, ×400. A nuclear protein, proliferating cell nuclear Ag, was used as a loading control. C, Heat shock induces Hsp72 expression and nuclear translocation in macrophages cultures. RAW 264.7 cells subject to mild heat shock (42.5°C, 1 h), and nuclear Hsp72 content was determined by Western blotting. D, Overexpression of Hsp72 attenuates H2O2-induced HMGB1 cytoplasmic translocation. RAW 264.7 cells transfected with empty pcDNA3.1 plasmid, or pcDNA3.1-Hsp72 construct were stimulated with H2O2 (0.125 mM) for 12 h, and the subcellular localization of HMGB1 was determined by immunocytochemical analysis (D, left). Relative fluorescence intensity of HMGB1 in the nuclear (N) and cytoplasmic (C) regions of multiple representative cells was assayed using the ImageProPlus software (D, right). Image is representative of three experiments with similar results. Red, HMGB1; blue, nuclei. Original magnification, ×1000. Values are means ± SEM (n = 50) of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Over Expression, Translocation Assay, Transfection, Immunocytochemistry, Cell Fractionation, Western Blot, Control, Expressing, Plasmid Preparation, Construct, Fluorescence, Software

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Hsp72 and HMGB1 are coimmunoprecipitated after oxidative stress or heat shock. A, Coimmunoprecipitation of Hsp72 and HMGB1 from lysate of RAW 264.7 cells. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 construct were stimulated with H2O2 (0.125 mM) for 3 h, and whole-cell lysate was immunoprecipitated with various Abs: nonspecific rabbit serum (Sr), nonspecific mouse serum (Sm), or Abs specific for Hsp72 or HMGB1, respectively. In parallel experiment, RAW 264.7 cells were subjected to mild heat shock (42.5°C for 1 h) and returned to 37°C for 6 h, and whole-cell lysate was immunoprecipitated with HMGB1- or Hsp72-specific Abs, respectively. The precipitated complexes were separately immunoblotted with Hsp72- or HMGB1-specific Abs. IP, immunoprecipitation; IB, Immunoblotting. Hsp72-transfected cell lysate (L) was used as a positive control. Blot is representative of three experiments with similar results. B, Coimmunoprecipitation of Hsp72 and HMGB1 from lysate of human leukemia K562 cells. K562 cells were subjected to oxidative stress (by stimulating withH2O2, 0.125 mM, for 12 h) or mild heat shock (HS, 42.5°C, 1 h, and then returned to 37°C for 6 h), and whole-cell lysate was immunoprecipitated with HMGB1-specific Abs. The precipitated complexes were then sequentially blotted with Hsp72- or HMGB1-specific Abs, respectively. IP, immunoprecipitation; IB, immunoblotting; Ctrl, control cells. Blot is representative of two experiments with similar results. C, Coimmunoprecipitation of Hsp72 and HMGB1 from nuclear and cytoplasmic fraction of RAW 264.7 cells transfected with pcDNA3.1-Hsp72. Cells were subjected to oxidative stress (H2O2, 0.125 mM for 3 h; H), or mild heat shock (42.5°C for 1 h, then returned to 37°C for 6 h; HS), and nuclear or cytoplasmic proteins were immunoprecipitated with Hsp72-or HMGB1-specific Abs. The precipitated complexes were then blotted with Hsp72- or HMGB1-specific Abs. Hsp72-transfected cell lysates was used as a positive control. C, control cells. Blot is representative of two experiments with similar results. D, Stoichiometry analysis of Hsp72-bound HMGB1 in nuclear fractions. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 were stimulated with H2O2 (0.125 mM) for 3 h, and levels of HMGB1 in the whole-cell extracts before (1, set at 100%) or after (2) immunoprecipitation with excessive amount of Hsp72-specific Ab was determined by Western blotting. Blot is representative of three experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Positive Control, Control

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: An herb-derived antioxidant, quercetin, attenuates H2O2-induced Hsp72 nuclear translocation, and Hsp72-HMGB1 interaction in macrophage cultures. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 construct were pretreated with quercetin (50 μM) for 4 h and subsequently stimulated with H2O2 (0.125 mM) for 3 h. Subcellular localization of Hsp72 in cells was determined by immunocytochemistry (A) or Western blot (B). Results are representative of three experiments with similar results. Green, Hsp72; blue, nuclei. Original magnification, ×400). Ctrl, control cells; Q, + quercetin; H, + H2O2; QH, + quercetin + H2O2. In parallel experiments, whole-cell lysate was immunoprecipitated with Hsp72-specific Abs, and the precipitated complexes were immunoblotted with Hsp72- or HMGB1-specific Abs, respectively (C). IP, immunoprecipitation; IB, immunoblotting. Blot is representative of three experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Derivative Assay, Translocation Assay, Transfection, Construct, Immunocytochemistry, Western Blot, Control, Immunoprecipitation

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Identification of functional domains of Hsp72 for interacting with nuclear HMGB1. A, Schematic diagram of Hsp72 and two mutants lacking the NLS or PBD. B, Detection of HMGB1 and Myc-tagged Hsp72, Hsp72-ΔNLS, or Hsp72-ΔPBD by Western blotting. RAW 264.7 cells were transiently transfected with pcDNA3.1-Hsp72, pcDNA3.1-Hsp72-ΔNLS, or pcDNA3.1-Hsp72-ΔPBD and stimulated with H2O2 (0.125 mM) for 3 h; nuclear extract was assayed for HMGB1 and Myc-tagged proteins by Western blotting (Immunoblotting; IB) analysis. Blots are representative of two independent experiments with similar results. C, Coimmunoprecipitation of HMGB1 with Myc-tagged Hsp72, Hsp72-ΔNLS, or Hsp72-ΔPBD. In parallel experiments, nuclear extracts were immunoprecipitated (IP) with HMGB1-specific Abs, and the precipitated complexes were then assayed for levels of HMGB1 or Myc-tagged proteins by Western blotting. Blots are representative of two independent experiments with similar results. D, Western blotting of H2O2-induced HMGB1 release (D, left) and translocation (D, right) in RAW 264.7 cells transfected with empty plasmid, pcDNA3.1-Hsp72, pcDNA3.1-Hsp72-ΔNLS, or pcDNA3.1-Hsp72-ΔPBD. Tubulin was used as a loading control. Blots are representative of three independent experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Functional Assay, Western Blot, Transfection, Immunoprecipitation, Translocation Assay, Plasmid Preparation, Control

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Hypothetical role of Hsp72 in the regulation of oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to stresses (e.g., heat shock), Hsp72 is produced to maintain a pool of Hsp72 in the cytoplasm. Upon stimulation with secondary oxidative stress (e.g., H2O2), Hsp72 is translocated into the nucleus, where it directly, or indirectly, interacts with various nuclear proteins (such as HMGB1 and histone deacetylase 1 (HDAC1). The intranuclear Hsp72-HMGB1 consequently prevents HMGB1 cytoplasmic translocation and subsequent release via the secretory lysosome pathway.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Translocation Assay, Produced, Histone Deacetylase Assay

Journal: Cancer science

Article Title: SNRPB2 promotes triple-negative breast cancer progression by controlling alternative splicing of MDM4 pre-mRNA.

doi: 10.1111/cas.16356

Figure Lengend Snippet: FIGURE 4 Effects of SNRPB2 on alternative splicing (AS) in triple-negative breast cancer cells. (A) Pie plot of five types of significantly altered AS events (siSNRPB2 vs. siControl [siCtr]) in MDA-MB-231-LM2 cells according to the rMATS analysis. (B) Percentage of inclusion and exclusion of exons or introns in five types of significantly altered AS events (siSNRPB2 vs. siCtr) in MDA-MB-231-LM2 cells. (C) Volcano plot showing significantly altered AS events (siSNRPB2 vs. siCtr) in MDA-MB-231-LM2 cells. MDM4 exon 6 was the most significant skipping exon in the siSNRPB2 group compared with the siCtr group. (D) Distribution of significantly altered AS events in protein-coding and noncoding transcripts. (E) Distribution of significantly altered AS events in coding sequence (CDS), 5′-UTR, 3′-UTR, promoter, and other regions of the transcripts. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of genes with significantly altered AS events (siSNRPB2 vs. siCtr). (G, H) RT-PCR and electrophoresis of (G) RBX1 exon 4 and (H) FOXM1 exon 6 expression in MDA-MB-231 and SUM159 cells with transfection of Control siRNA or SNRPB2 siRNAs. (I) Abundance of RBX1 transcripts in normal tissues and breast tumors from The Cancer Genome Atlas breast cancer (TCGA-BRCA) dataset was analyzed. Schemes of the most abundant RBX1 mRNA sequences with alternative splicing exons. Exon 4 skipping of RBX1 would lead to a frameshift and produce a novel protein 88 aa in length. (J) Western blot detection of RBX1 protein levels in MDA-MB-231 and SUM159 cells with transfection of Control siRNA or SNRPB2 siRNAs. (K) Abundance of FOXM1 transcripts in normal tissues and breast tumors from the TCGA-BRCA dataset was analyzed. Schemes of the most abundant FOXM1 mRNA sequences with alternative splicing exons. Exon 6 skipping of FOXM1 generates a short transcript and produces a short protein (748 aa). (L) Western blot detection of FOXM1 protein levels in MDA-MB-231 and SUM159 cells with transfection of Control siRNA or SNRPB2 siRNAs. *p < 0.05. A3SS, alternative 3′-splice site; A5SS, alternative 5′-splice site; FDR, false discover rate; IR, intron retention; MXE, mutually exclusive exon; PSI, percent spliced in; SE, skipped exon.

Article Snippet: The information of Abs was as follows: anti- SNRPB2 (#13512- 1- AP, Proteintech, 1:1000), anti- β- actin (#66009- 1- Ig, Proteintech, 1:5000), anti- MCM2 (#10513- 1- AP, Proteintech, 1:1000), antiCDC20 (#AF6456, Beyotime, 1:1000), anti- MDM4 (#28747- 1- AP, Proteintech, 1:1000), anti- RBX1 (#14895- 1- AP, Proteintech, 1:1000), anti- FOXM1 (#13147- 1- AP, Proteintech, 1:1000), HRP- conjugated goat anti- rabbit (#SA00001- 2, Proteintech, 1:10000), and HRPconjugated goat anti- mouse (#SA00001- 1, Proteintech, 1:10000).

Techniques: Alternative Splicing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Expressing, Transfection, Control, Western Blot

Journal: Materials Today Bio

Article Title: Simultaneous targeting and suppression of heat shock protein 60 to overcome heat resistance and induce mitochondrial death of tumor cells in photothermal immunotherapy

doi: 10.1016/j.mtbio.2024.101282

Figure Lengend Snippet: Activation effects of DAMPs of 4T1 cells treated differently on DCs. The amounts of (A) ATP, (B) HSP60, and (C) HMGB1 released by the 4T1 cells quantified by ELISA, and (D) exposed CRT on the 4T1 cell membrane detected by flow cytometry. (E) and (F): Mean fluorescence intensity of MHCII and CD86 for the DC2.4 cells co-incubated with the supernatant of 4T1 cell treated differently, respectively (n = 3).

Article Snippet: Beyotime biotechnology provided the adenosine 5′-triphosphate (ATP) bioluminescent assay kit and cytochrome C (Cyto C) antibody, and Chondrex provided the high mobility group box 1 (HMGB1) detection kit.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Membrane, Flow Cytometry, Fluorescence, Incubation