Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Application of 18 β -glycyrrhetinic acid Fluorescent probes in cell imaging

doi: 10.1080/14756366.2026.2631869

Figure Lengend Snippet: Effects of 18 β -GA-PAL-ABP on LPS-induced release of HDAC8, STAT3, P-STAT3, and SOCS3. A: expression of HDAC8 protein; B: expression of STAT3 protein; C: expression of P-STAT3 protein; and D: expression of SOCS3 protein. E: The protein expression map in RAW 264.7 cells treated with Class I compounds and drug concentrations; F: The protein expression map in RAW 264.7 cells treated with Class II compounds and drug concentrations; G: The protein expression map in RAW 264.7 cells treated with Class III compounds and drug concentrations. Compared to the Control group, # p < 0.05, ## p < 0.01, ### p < 0.001. Compared to the Model group, * p < 0.05, ** p < 0.01, *** p < 0.001. Compared to the 18β-GA group, + p < 0.05, ++ p < 0.01, +++ p < 0.001.

Article Snippet: GAPDH (8884, CST, 1:5000), HDAC8 (#66042, CST, 1:1500), P-STAT3 (#9154, CST, 1:1500), STAT3 (#12640, CST, 1:1000), SOCS3 (#52113, CST, 1:2000), SDS-PAGE reagents (M00657, Genescript, Nanjing, China).

Techniques: Expressing, Control

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: His6-tagged HDAC8 (His6-HDAC8) proteins (Ag11692) 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: His6-tagged HDAC8 (His6-HDAC8) proteins (Ag11692) 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

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: K96 and K294 are the acetylation sites of β-TrCP1 that are regulated by Tip60 and HDAC8 and are responsible for hypoxia-induced β-TrCP1 degradation. ( A ) LC–MS/MS analysis of Flag-tagged β-TrCP1 identified K96, K220, and K294 as potential β-TrCP1 acetylation sites. MS spectra of the peptides containing the three acetylation sites are shown. ( B ) Prediction of potential acetylation sites of β-TrCP1 using the GPS-PAIL database ( http://pail.biocuckoo.org/ ). ( C ) HEK293T cells transfected with Flag-tagged wild-type (WT) β-TrCP1 or seven lysine (K) to alanine (A)-mutated β-TrCP1 were challenged with 1% O 2 for 24 h (Hyp) or not (Nor) combined with (+) or without (-) 10 μM TSA for 12 h as indicated. WB assays were performed with the indicated antibodies. ( D ) HEK293T cells were transfected with Flag-tagged wild-type (WT) β-TrCP1 or K96A- or K294A-mutated β-TrCP1 for 48 h under normoxic conditions. Co-IP assays were performed with anti-Flag antibodies, followed by WB analyses. Co-IP with IgG was performed as a negative control. ( E ) Alignment of the K96- and K294-containing peptide sequences from distinct species. ( F ) Flag-tagged wild-type (WT) β-TrCP1 or K96A- or K294A-mutated β-TrCP1 combined with (+) or without (-) HA-tagged Tip60 (HA-Tip60) as indicated was transfected into HEK293T cells for 48 h under normoxic conditions. Co-IP assays were performed with anti-Flag antibodies, followed by WB analyses. Co-IP with IgG was performed as a negative control. ( G ) Flag-tagged wild-type (WT) β-TrCP1 or K96A- or K294A-mutated β-TrCP1 combined with (+) or without (-) HDAC8-specific siRNA (siHDAC8) as indicated was transfected into HEK293T cells for 48 h under normoxic conditions. Co-IP assays were performed with anti-Flag antibodies, followed by WB analyses. Co-IP with IgG was performed as a negative control. ( H ) HEK293T cells were transfected with Flag-tagged wild-type β-TrCP1 (Flag-β-TrCP1-WT) or K96A- or K294A-mutated β-TrCP1 (Flag-β-TrCP1-K96A and Flag-β-TrCP1-K294A) 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. ( I ) K to glutamine (Q) mutations at the K96 and K294 sites of β-TrCP1 were generated. HEK293T cells were transfected with Flag-β-TrCP1-WT, Flag-tagged K96Q- or K294Q-mutated β-TrCP1 (Flag-β-TrCP1-K96Q and Flag-β-TrCP1-K294Q) 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. ( J ) HEK293T cells were transfected with WT or K96A-, K294A-, K96Q-, or K294Q-mutated Flag-β-TrCP1 for 48 h. Cells were then cultured under hypoxic conditions (Hyp) for 24 h combined with 25 μM MG132 treatment for 12 h. Cells were subsequently lysed under denaturing conditions, and β-TrCP1 was immunoprecipitated (IP) with anti-Flag antibodies. IP with IgG was performed as a negative control. The precipitated proteins were subjected to WB analysis. ( K ) HEK293T cells transfected with WT or K96A-, K294A-, K96Q-, or K294Q-, or double KA- or KQ-mutated Flag-β-TrCP1 were challenged with 1% O 2 for 24 h (Hyp) or not (Nor) as indicated. WB assays were performed to measure the levels of the indicated proteins. ( L ) WT or K96A-, K2 22 94A-, or double KA-mutated Flag-β-TrCP1 together with (+) or without (-) siHDAC8 were transfected into HEK293T cells. Cells were then challenged with 1% O 2 for 24 h (Hyp) or not (Nor) as indicated. WB assays were performed to measure the levels of the indicated proteins. ( M ) WT or seven KA-mutated Flag-β-TrCP1 together with (+) or without (-) HA-Tip60 were transfected into HEK293T cells under normoxic conditions. WB assays were performed to measure the levels of the indicated proteins. ( N) WT or K96A- or K294A-mutated Flag-β-TrCP1 together with (+) or without (-) HA-Tip60 were transfected into HEK293T cells. Cells were then challenged with 1% O 2 for 24 h (Hyp). WB assays were performed to measure the levels of the indicated proteins. ( O ) Working model. Tip60 mainly regulates the acetylation of β-TrCP1 at K294, whereas HDAC8 can eliminate the acetylation of β-TrCP1 at both K96 and K294

Article Snippet: His6-tagged HDAC8 (His6-HDAC8) proteins (Ag11692) were purchased from Proteintech (China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Co-Immunoprecipitation Assay, Negative Control, Generated, Cell Culture, Immunoprecipitation

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: β-TrCP1 degradation is required for hypoxia-induced cell death and tissue injury. ( A ) HepG2 cells were transfected with siHDAC8 or siHDAC8 combined with siβ-TrCP1 or siCont. for 24 h. Cells were then exposed to 1% O 2 (Hyp) for the indicated times. Cell death was measured by PI staining assay. ( B ) HepG2 cells were transfected with Flag-Tip60 or Flag-Tip60 combined with siβ-TrCP1 or Flag empty vector for 24 h. Cells were then exposed to 1% O 2 (Hyp) for the indicated times. Cell death was measured by PI staining assay. ( C ) HepG2 cells were transfected with wild-type (WT) or K96Q- or K294Q-mutated Flag-β-TrCP1 for 24 h. Cells were then exposed to 1% O 2 (Hyp) for the indicated times. Cell death was measured by PI staining assay. ( D ) HepG2 cells were transfected with siSMURF2 or siSMUFR2 combined with siβ-TrCP1 or siCont. for 24 h. Cells were then exposed to 1% O 2 (Hyp) for the indicated times. Cell death was measured by PI staining assay. ( E ) HepG2 cells were transfected with Flag-β-TrCP1 or Flag empty vector for 24 h. Cells were then exposed to 1% O 2 for 72 h (Hyp) or not (Nor). WB assays were performed to measure the levels of the indicated proteins. ( F ) HepG2 cells were transfected with siβ-TrCP1 or siCont. for 24 h. Cells were then exposed to 1% O 2 for 72 h (Hyp) or not (Nor). WB assays were performed to measure the levels of the indicated proteins. ( G ) HepG2 cells were transfected with siβ-TrCP1, siβ-TrCP1 together with p53-specific siRNA (sip53), or siCont. for 24 h. Cells were then exposed to 1% O 2 (Hyp) for the indicated times or not (Nor). Cell death was measured by PI staining assay. ( H ) HepG2 cells were transfected with HA-tagged β-TrCP1 (HA-β-TrCP1), HA-β-TrCP1 together with Flag-tagged p53 (Flag-p53), or Flag empty vector (Cont.) for 24 h. Cells were then exposed to 1% O 2 (Hyp) for the indicated times or not (Nor). Cell death was measured by PI staining assay. ( I ) Wild-type (WT), Tip60-specific shRNA (shTip60)-containing AAV8-infected or shTip60-AAV8 combined with Flag-β-TrCP1-containing AAV8-infected mice were exposed to 7% O 2 for 72 h (Hyp) or not (Nor). Histological analyses of TUNEL, immunostaining for cleaved caspase 3 and H&E assays were performed on liver sections. DAPI staining was performed to detect the nuclei. ( J ) WT, shTip60-containing AAV8-infected or shTip60-AAV8 combined with Flag-β-TrCP1-containing AAV8-infected mice were exposed to Hyp for 72 h (+) or not (-) as indicated. Serum ALT and AST levels were measured ( n = 6). (K ) WT, β-TrCP1 −/− , and WT or β-TrCP1 −/− mice infected with HDAC8-specific shRNA (shHDAC8)-containing AAV8 were exposed to 7% O 2 for 72 h (Hyp) or not (Nor). Histological analyses of TUNEL, immunostaining for cleaved caspase 3 and H&E assays were performed on liver sections. DAPI staining was performed to detect the nuclei. ( L ) WT, β-TrCP1 −/− , and WT or β-TrCP1 −/− mice infected with shHDAC8-AAV8 were exposed to 7% O 2 for 72 h (Hyp) or not (Nor). Serum ALT and AST levels were measured ( n = 6). ( M ) WT, β-TrCP1 −/− and β-TrCP1 −/− mice infected with p53-specific shRNA (shp53)-containing AAV8 were exposed to 7% O 2 for 72 h (Hyp) or not (Nor). Histological analyses of TUNEL, immunostaining for cleaved caspase 3 and H&E assays were performed on liver sections. DAPI staining was performed to detect the nuclei. ( N ) WT, β-TrCP1 −/− or β-TrCP1 −/− mice infected with shp53-containing AAV8 were exposed to Hyp for 72 h (+) or not (-) as indicated. Serum ALT, AST and bilirubin levels were measured ( n = 6). ( O ) Working model. Under normoxic conditions, Tip60 is prolyl-hydroxylated and stabilized by PHD2, and then Tip60 acetylates β-TrCP1 to stabilize β-TrCP1, allowing cells with lower levels of p53. Under hypoxic conditions, impaired prolyl hydroxylation of Tip60 results in the degradation of Tip60, and HDAC8 is recruited to β-TrCP1, synergistically promoting a decrease in β-TrCP1 acetylation. Thus, more SMURF2 is recruited to β-TrCP1 and ubiquitinates β-TrCP1 to promote its degradation. Ultimately, p53 is accumulated, and cell death occurs. Data information: Bars and error bars represent mean ± SD, n = 4 independent repeats in ( A - D and H ), n = 3 independent repeats in ( G ), n = 6 independent repeats in ( J and L ). Two-tailed unpaired Student’s t test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: His6-tagged HDAC8 (His6-HDAC8) proteins (Ag11692) were purchased from Proteintech (China).

Techniques: Transfection, Staining, Plasmid Preparation, shRNA, Infection, TUNEL Assay, Immunostaining, Two Tailed Test