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. 2023 Aug 9;65:102838. doi: 10.1016/j.redox.2023.102838

Fig. 6.

Fig. 6

Cytoglobin regulates HMGB2 genome wide binding. (a) Schematic of the immunoprecipitation and mass spectrometry approach used to identify cytoglobin-interacting proteins in HEK293 cells stably expressing empty (pCMV6-tag) and hCYGB-tag vectors. (b) Venn diagram showing limited overlap of cytoglobin protein interactors between control and hydrogen peroxide treated samples. 54 proteins were interacting with CYGB before hydrogen peroxide addition and 31 proteins after hydrogen peroxide treatment. Subsequent analysis yielded several cytoglobin nuclear-associated proteins obtained from Co-IP MS/MS in hydrogen peroxide treated conditions. (c) Representative immunofluorescence images of proximity ligation assay for CYGB and HMGB2 in HEK293 cells with or without stable expression of cytoglobin (hCYGB); scale bar, 30 μm. (d) Quantitation of proximity ligation assay signal (number of nuclear particles per nuclei) of HEK293 cells stabling expressing empty vector (pCDNA) and human cytoglobin (hCYGB) following hydrogen peroxide treatment for 10 min. (e) HMGB2 ChiP-seq in pCDNA and hCYGB HEK 293 cells with or without treatment with hydrogen peroxide showing increase in HGMB2-genome wide binding in the presence of cytoglobin and loss of binding after treatment with hydrogen peroxide. Right panel: Peak position (in % peaks) of the ChiP analysis of HMGB2 (f) Western blot analysis of lysates from pCDNA (empty vector) and human cytoglobin (hCYGB) with and without hydrogen peroxide treatment probed for HMGB2 (24 kDa and β-Actin (ACTB, loading control, 40 kDa). (g) Cytoglobin heterodimerization is associated with an increase in HMGB2 genome-wide binding. Hydrogen peroxide (H2O2) increases cytoglobin-HMGB2 heterodimerization but inhibits HMGB2 genome-wide binding. (h) Quantitative analysis of HMGB2 mean fluorescence intensity (MFI) following HMGB2 silencing with siRNA targeting HMGB2 (SiHMGB2) or scrambled siRNAs (SCR); data are presented as±SEM; p values were determined by unpaired Student's t-test. (i) HMGB2 was silenced in pCDNA (empty vector) and hCYGB (CYGB expressing) HEK cells, treated with 200 μM bolus hydrogen peroxide, and quantitated for %cells positive with γ-H2AX. Data are presented as±SEM; p values were determined by two-way ANOVA followed by Tukey's post hoc test for multiple comparisons.