Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Tip60-HDAC8-SMURF2-mediated β-TrCP1 degradation is a key mechanism for hypoxia-induced cell death and tissue injury

doi: 10.1007/s00018-025-05983-4

Figure Lengend Snippet: HDAC8 is the deacetylase of β-TrCP1 and is essential for hypoxia-induced β-TrCP1 degradation. ( A ) HEK293T and HepG2 cells were treated with 10 mM NAM, 10 μM TSA, 25 μM MG132, or DMSO as a control for 12 h. WB assays were performed to measure the levels of the indicated proteins. ( B ) Flag-tagged β-TrCP1 (Flag-β-TrCP1)-transfected HEK293T and HepG2 cells were exposed to 1% O 2 for 24 h (Hyp) or not (Nor). Co-IP assays were performed with anti-Flag antibodies, followed by WB analyses. Co-IP with IgG was performed as a negative control. ( C ) Flag-β-TrCP1-transfected HEK293T cells were exposed to 1% O 2 for 24 h (Hyp) or not (Nor) combined with 10 μM TSA treatment for 12 h. Co-IP assays were performed with anti-Flag antibodies, followed by WB analyses. Co-IP with IgG was performed as a negative control. ( D ) siRNAs specific for distinct HDACs (siHDACs) or control siRNA (siCont.)-transfected HEK293T cells were challenged with 1% O 2 for 24 h (Hyp) or not (Nor). WB assays were performed to measure the levels of the indicated proteins. ( E ) Flag-β-TrCP1-transfected HEK293T cells were exposed to 1% O 2 for 24 h (Hyp) or not (Nor). Co-IP assays were performed with anti-Flag antibodies followed by WB analyses. Co-IP with IgG was performed as a negative control. ( F ) Flag-β-TrCP1-transfected HEK293T cells were treated with 10 μM TSA for 12 h. The Flag-β-TrCP1 proteins were immunoprecipitated with anti-Flag antibodies. The precipitated Flag-β-TrCP1 was recognized as acetylated Flag-β-TrCP1 (Ac-Flag-β-TrCP1). In vitro deacetylation assays were performed with the precipitated Flag-β-TrCP1 and the purified HDAC8, HDAC7, or HDAC4 proteins incubated with or without 10 μM TSA. WB assays were performed with the indicated antibodies. ( G ) GST-tagged β-TrCP1 (GST-β-TrCP1) and His6-tagged HDAC8 (His6-HDAC8) were purified from E. coli (left) and coincubated. IP analysis with antibodies against β-TrCP1 or HDAC8 was performed (right two panels). ( H ) HEK293T cells were transfected with Flag-β-TrCP1 combined with HDAC8-specific siRNA (siHDAC8) or siCont. for 24 h. Cells were then cultured under normoxic or hypoxic conditions for another 24 h. Co-IP assays were performed with anti-Flag antibodies followed by WB analyses. Co-IP with IgG was performed as a negative control. ( I ) HEK293T cells were transfected with increasing amounts of Flag-tagged HDAC8 (Flag-HDAC8) for 48 h under normoxic conditions. Cells were then treated with or without 10 μM TSA or 25 μM MG132 for another 12 h. WB assays were performed to measure the levels of the indicated proteins. ( J ) HEK293T cells were transfected with siHDAC8 or siCont. for 48 h under normoxic conditions. Cells were then treated with or without 25 μM MG132 for another 12 h. WB assays were performed to measure the levels of the indicated proteins. ( K ) HEK293T cells were transfected with Flag-HDAC8 or Flag empty vector for 48 h under normoxic conditions. Chase assays were performed by treating cells with 50 μg/mL CHX for the indicated times. WB assays were performed to measure the levels of the indicated proteins. ( L ) HEK293T cells were transfected with siHDAC8 or siCont. for 48 h under normoxic conditions. Chase assays were performed by treating cells with 50 μg/mL CHX for the indicated times. WB assays were performed to measure the levels of the indicated proteins. ( M ) Working model. HDAC8-mediated deacetylation of β-TrCP1 is essential for hypoxia-induced β-TrCP1 degradation. Data information: Bars and error bars represent mean ± SD, n = 3 independent repeats. Two-tailed unpaired Student’s t test was performed. * p < 0.05; ** p < 0.01

Article Snippet: Antibodies against SLC7A11 (26864-1-AP), HA-tag (51064-2-AP), GST-tag (66001-2-Ig), His6-tag (66005-1-Ig) and Myc-tag (60003-2-Ig) were purchased from Proteintech (China).

Techniques: Histone Deacetylase Assay, Control, Transfection, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, In Vitro, Purification, Incubation, Cell Culture, Plasmid Preparation, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Tip60-HDAC8-SMURF2-mediated β-TrCP1 degradation is a key mechanism for hypoxia-induced cell death and tissue injury

doi: 10.1007/s00018-025-05983-4

Figure Lengend Snippet: Tip60 is the acetyltransferase of β-TrCP1, and Tip60-mediated β-TrCP1 acetylation is impaired upon hypoxia. ( A ) HEK293T cells were transfected with multiple HA- or Flag-tagged acetyltransferases or HA empty vector as indicated for 48 h under normoxic conditions. WB assays were performed to measure the levels of the indicated proteins. ( B ) HEK293T cells were transfected with Flag-tagged wild-type Tip60 (Flag-Tip60), the dominant negative (DN) mutant lacking the enzymatic activity of Tip60 (Flag-Tip60-DN), or Flag empty vector for 48 h under normoxic conditions. Cells were then treated with or without 30 μM MG149 for another 12 h. WB assays were performed to measure the levels of the indicated proteins. ( C ) HEK293T cells were transfected with Flag-Tip60, Flag-Tip60-DN, or Flag empty vector for 48 h. Cells were then challenged with 1% O 2 for another 24 h (Hyp) or not (Nor). WB assays were performed to measure the levels of the indicated proteins. ( D ) HEK293T cells were cultured under normoxic or hypoxic conditions for 24 h. WB assays were performed to measure the levels of the indicated proteins. ( E ) HEK293T cells were cultured under normoxic conditions. Co-IP assays were performed with anti-β-TrCP1 or anti-Tip60 antibodies, followed by WB analyses to measure the levels of the indicated proteins. Co-IP with IgG was performed as a negative control. ( F ) HEK293T cells were exposed to 1% O 2 for 24 h (Hyp). Co-IP assays were performed with anti-β-TrCP1 or anti-Tip60 antibodies, followed by WB analyses to measure the levels of the indicated proteins. Co-IP with IgG was performed as a negative control. ( G ) HEK293T cells were exposed to 1% O 2 for 24 h (Hyp) or not (Nor). Cells were then treated with or without 25 μM MG132 for another 12 h. Co-IP assays were performed with anti-β-TrCP1 antibodies followed by WB analyses to measure the levels of the indicated proteins. Co-IP with IgG was performed as a negative control. ( H ) GST-tagged Tip60 (GST-Tip60) and His6-tagged β-TrCP1 (His6-β-TrCP1) were purified from E. coli (left) and coincubated. His6 or GST pull-down analysis with Ni-beads or Sepharose 4B-beads was performed (right two panels). ( I ) In vitro acetylation assays were performed by incubating GST-Tip60 and His6-β-TrCP1 with or without acetyl coenzyme A (Ac-CoA). WB assays were performed with the indicated antibodies. ( J ) HEK293T cells transfected with HA-tagged β-TrCP1 (HA-β-TrCP1) together with Flag-Tip60 or Flag-Tip60-DN were challenged with 1% O 2 for 24 h or not. Co-IP assays were performed with anti-HA antibodies, followed by WB analyses. Co-IP with IgG was performed as a negative control. ( K ) HEK293T cells transfected with Flag-tagged β-TrCP1 (Flag-β-TrCP1) together with Tip60-specific siRNA (siTip60: (+)) or control siRNA (siTip60: (-)) were challenged with 1% O 2 for 24 h or not. Co-IP assays were performed with anti-Flag antibodies followed by WB analyses. Co-IP with IgG was performed as a negative control. ( L ) HEK293T cells were transfected with siTip60 or siCont. for 48 h. Cells were then treated with or without 25 μM MG132 and cultured under normoxic or hypoxic conditions for another 12 h. WB assays were performed to measure the levels of the indicated proteins. ( M ) HEK293T cells were transfected with Flag-Tip60 or Flag empty vector for 48 h under normoxic conditions. Chase assays were performed by treating cells with 50 μg/mL CHX for the indicated times. WB assays were performed to measure the levels of the indicated proteins. ( N ) HEK293T cells were transfected with siTip60 or siCont. for 48 h under normoxic conditions. Chase assays were performed by treating cells with 50 μg/mL CHX for the indicated times. WB assays were performed to measure the levels of the indicated proteins. ( O ) Working model. Tip60 is the acetyltransferase of β-TrCP1, and Tip60-mediated β-TrCP1 acetylation is impaired upon hypoxia

Article Snippet: Antibodies against SLC7A11 (26864-1-AP), HA-tag (51064-2-AP), GST-tag (66001-2-Ig), His6-tag (66005-1-Ig) and Myc-tag (60003-2-Ig) were purchased from Proteintech (China).

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Activity Assay, Cell Culture, Co-Immunoprecipitation Assay, Negative Control, Purification, In Vitro, Control