Journal: Toxicology letters

Article Title: Manganese phosphorylates Yin Yang 1 at serine residues to repress EAAT2 in human H4 astrocytes.

doi: 10.1016/j.toxlet.2021.11.007

Figure Lengend Snippet: Fig.1. Mn phosphorylates YY1 at serine residues via AurkB and CK2 in H4 astrocytes. (A) Astrocytes were pre-treated with the AurkB inhibitor Hesperadin (Hesp, 250 nM,1 h), then exposed to Mn (250 mM, 3 h) and immunoprecipitated with YY1 antibody, followed by western blotting for phosphoserine. (B) Astrocytes were pre-treated with the CK2 inhibitor CX-4945 (CX, 10 mM, 1 h), then exposed to Mn (250 mM, 3 h) and immunoprecipitated for YY1, followed by western blotting for phosphoserine. (**p < 0.01, ***p < 0.001, compared to the control; #p < 0.05, ##p < 0.01, ANOVA followed by Tukey’s post hoc test; N = 3).

Article Snippet: Antibodies for YY1 (sc-7341), HDAC1 (sc-81598), HDAC3 (sc-376957), β-actin (sc-47778), and histone H4 (sc-25260) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Toxicology letters

Article Title: Manganese phosphorylates Yin Yang 1 at serine residues to repress EAAT2 in human H4 astrocytes.

doi: 10.1016/j.toxlet.2021.11.007

Figure Lengend Snippet: Fig. 2. Mn-induced YY1 phosphorylation via AurkB and CK2 leads to YY1 nuclear translocation. (A) Astrocytes were pre-treated with Hesp (250 nM, 1 h) and exposed to Mn (250 mM, 3 h), then cells were fractionated and analyzed for YY1 protein levels in the nuclear fraction by western blotting. Nuclear protein histone H4 was used as a loading control. (B) Astrocytes were pre-treated with CX (10 mM, 1 h) and exposed to Mn (250 mM, 3 h), then cells were fractionated and analyzed for YY1 protein expression in the nuclear fraction by western blotting. (C) Astrocytes were pre-treated with Hesp or CX and exposed to Mn for the indicated time periods, then analyzed via immunocytochemistry (ICC) for YY1 nuclear expression. Scale: 0-25 mM. (*p < 0.05, ***p < 0.001, compared to the control; #p < 0.05, ##p < 0.01, ANOVA followed by Tukey’s post hoc test; N = 3).

Article Snippet: Antibodies for YY1 (sc-7341), HDAC1 (sc-81598), HDAC3 (sc-376957), β-actin (sc-47778), and histone H4 (sc-25260) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Control, Expressing, Immunocytochemistry

Journal: Toxicology letters

Article Title: Manganese phosphorylates Yin Yang 1 at serine residues to repress EAAT2 in human H4 astrocytes.

doi: 10.1016/j.toxlet.2021.11.007

Figure Lengend Snippet: Fig. 3. Mn-induced YY1 phosphorylation increases interaction between YY1 and HDACs in H4 astrocytes. (A) Astrocytes were pre-treated with Hesp (250 nM, 1 h), then exposed to Mn (250 mM, 3 h) and immunoprecipitated for YY1, followed by western blotting for HDAC1. (B) Astrocytes were pre-treated with CX (10 mM, 1 h), then exposed to Mn (250 mM, 3 h) and co-immunoprecipitated for YY1/HDAC3 interaction. (*p < 0.05, **p < 0.01, ***p < 0.001, compared to the control; #p < 0.05, ANOVA followed by Tukey’s post hoc test; N = 3).

Article Snippet: Antibodies for YY1 (sc-7341), HDAC1 (sc-81598), HDAC3 (sc-376957), β-actin (sc-47778), and histone H4 (sc-25260) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Control

Journal: Toxicology letters

Article Title: Manganese phosphorylates Yin Yang 1 at serine residues to repress EAAT2 in human H4 astrocytes.

doi: 10.1016/j.toxlet.2021.11.007

Figure Lengend Snippet: Fig. 4. Mn-induced YY1 phosphorylation via AurkB and CK2 increases YY1 binding to the EAAT2 promoter in H4 astrocytes. (A) Astrocytes were pre-treated with Hesp (250 nM, 1 h) and exposed to Mn (250 mM, 6 h), then cells were fractionated and the DAPA assay was performed to assess YY1 binding to the EAAT2 promoter. (B) Astrocytes were treated with Hesp and Mn for the indicated time periods, followed by the ChIP assay to assess YY1 binding to EAAT2 promoter. (C) Astrocytes were pre-treated with CX (10 mM,1 h) and exposed to Mn (250 mM, 6 h), then cells were fractionated and the DAPA assay was performed. (D) Astrocytes were treated with CX and Mn for the indicated time periods and the ChIP assay was performed. (*p < 0.05, **p < 0.01, ***p < 0.001, compared to the control; #p < 0.05, ##p < 0.01, ANOVA followed by Tukey’s post hoc test; N = 3).

Article Snippet: Antibodies for YY1 (sc-7341), HDAC1 (sc-81598), HDAC3 (sc-376957), β-actin (sc-47778), and histone H4 (sc-25260) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Binding Assay, Control