Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel benzohydroxamate-based histone deacetylase 6 (HDAC6) inhibitors with the ability to potentiate anti-PD-L1 immunotherapy in melanoma

doi: 10.1080/14756366.2023.2201408

Figure Lengend Snippet: HDAC-inhibitory activity and isoform-selectivity of the newly synthesised compounds.

Article Snippet: The HDAC3 enzyme (BML-SE515-0050) and HDAC8 enzyme (H90-30H-05) were bought from ENZO Inc. (New York, NY) and SignalChem (Richmond, Canada), respectively.

Techniques: Activity Assay

Journal: Cell chemical biology

Article Title: HDAC3 and HDAC8 PROTAC dual degrader reveals roles of histone acetylation in gene regulation

doi: 10.1016/j.chembiol.2023.07.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-HDAC8 , Proteintech , Cat# 17548–1-AP, RRID: AB_2116926.

Techniques: Recombinant, Software

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: a , b HDAC8 Western blot on wild type (WT) adult mouse ( a ) whole sciatic nerve lysates or ( b ) after subcellular fractionation of cytoplasmic (C) and nuclear (N) fractions and quantification normalized to GAPDH ( a ), at 1, 3, 5, 12 and 30 dpl in crushed (Cr) compared to contralateral (Co) sciatic nerves. ( b ) GAPDH and Lamin A/C = markers of cytoplasmic and nuclear fractions, respectively. ( a ) N = 4 (1, 3, 5, 12 dpl) or 5 (30 dpl) animals per group, ( b ) 3 WT mice per time point, representative images are shown. c Semithin cross-sections of HDAC8 KO (H8KO) and Control (Ctrl) adult mouse sciatic nerves at 5 dpl and graphs showing the number of intact myelin rings per mm 2 (3 sections per animal). N = 3 animals per group. Turquoise arrows = intact myelin rings. d Semithin (upper panel) and ultrathin (lower panel) cross-sections of H8KO and Ctrl adult mouse sciatic nerves at 12 dpl and graphs showing the number of degenerated myelin rings per mm 2 , the percentage of remyelinated axons, the number of axons with diameter ≥2 µm per mm 2 , and the number of axons in regenerating clusters (diameter <2 µm per mm 2 ). N = 4 animals per group (3 semithin sections per animal, 5 images of 3 ultrathin sections per animal). Upper panel: magenta arrows = degenerated myelin rings. Lower panel: blue arrows = remyelinated axons, areas delineated by pink line = regenerating axon clusters. e Graphs showing the performance of H8KO and Ctrl mice at the Toe pinch, von Frey, Rotarod and Inverted grid tests (total number of steps per animal: 17 to 136) before or after sciatic nerve crush lesion. N (Toe pinch, von Frey) = 12, n (Rotarod, Inverted grid) = 7 (after lesion) or 12 (before lesion) animals per group. Paired ( a ) or unpaired ( c , d , e ) two-tailed (black asterisks) or one-tailed (n.s.) Student’s t-tests or two-way mixed ANOVA followed by post-hoc t-tests (von Frey), p values: *<0.05, **<0.01, ***<0.001, n.s. = non-significant, values = mean, error bars = s.e.m. Source data are provided as a Source data file.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques: Western Blot, Fractionation, Control, Two Tailed Test, One-tailed Test

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: a, b Whole sciatic nerve immunofluorescence (IF) of ( a ) Stathmin-2 (green) or ( b ) GAP43 (magenta), and DAPI labeling (blue, nuclei) and quantification of axonal regrowth at 3 dpl in HDAC8 KO (H8KO) nerves compared to Control (Ctrl) nerves. N = 4 animals per group (average length: 43 to 79 regrowing axons per animal). c Co-immunofluorescence (z-stacks projetions) of HDAC8 (green), Stathmin-2 (red) and Neurofilament (NF, magenta) and DAPI labeling (blue) in sciatic nerve cross-sections of wild type mice at 1 dpl (proximal part). Sections of 3 animals analyzed, a representative image is shown. White arrows = HDAC8-positive SCs surrounding Stathmin-2-positive axons, blue arrows = HDAC8-positive axons (NF-positive). The graph shows the percentage of HDAC8-positive SCs surrounding Stathmin-2-positive and Stathmin-2-negative axons. N = 3 animals per group, 25 to 94 HDAC8-positive SCs per animal. Unpaired two-tailed (black asterisks) or one-tailed (gray asterisks or n.s.) Student’s t-tests, p values: *<0.05, **<0.01, ***<0.001, n.s. = non-significant, values = mean, error bars = s.e.m. Source data are provided as a Source data file.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques: Immunofluorescence, Labeling, Control, Two Tailed Test, One-tailed Test

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: a c-Jun Western blot and quantification normalized to GAPDH at 1, 3, 5 and 12 dpl in crushed sciatic nerves of HDAC8 KO compared to Control mice. N = 9 (1 dpl) or 4 (3, 5, and 12 dpl) animals per group. b Co-immunofluorescence of c-Jun (green, upper panels) or phospho-c-Jun (p-c-Jun, green, lower panels) and F4/80 (magenta, macrophage marker), and DAPI labeling (blue, nuclei) in longitudinal sections of crushed and contralateral HDAC8 KO and Control sciatic nerves at 1 dpl. Three animals per group, representative images are shown. c HDAC8 and GAPDH (loading control) Western blots on lysates of rat SCs transduced with lentiviruses carrying an HDAC8-specific or a non-targeting control shRNA. Multiple (more than 3 times) independent experiments, representative images are shown. d – g Western blot of c-Jun ( d , f ) or phospho-c-Jun ( e , g ) in primary rat SCs cultured under conditions mimicking the conversion into the repair phenotype in hypoxia ( d , e : in hypoxic chamber for 16 h; f , g : CoCl 2 for 16 h), and quantification normalized to GAPDH in cells incubated with lentiviruses carrying an HDAC8 shRNA (H8sh) or control shRNA (Csh). Dashed lines indicate that samples were run on the same gel but not on consecutive lanes. N = 6 ( d , f , g ) or 5 ( e ) independent experiments per group. h Quantification of c-Jun mRNA levels by qRT-PCR in primary rat SCs cultured as above ( f, g ) and incubated with lentiviruses carrying an H8sh or Csh. N = 5 independent experiments per group. i Quantification of c-Jun promoter activity by luciferase gene reporter assay in cells cultured as above ( f, g, h ) and incubated with lentiviruses carrying an H8sh or Csh. N = 7 independent experiments per group. Paired ( a , d – g , i ) or unpaired ( h ) two-tailed (black asterisks) or one-tailed (gray asterisks). Student’s t-tests, p value: *<0.05, **<0.01, values = mean, error bars = s.e.m. Source data are provided as a Source data file.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques: Western Blot, Control, Immunofluorescence, Marker, Labeling, Transduction, shRNA, Cell Culture, Incubation, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Two Tailed Test, One-tailed Test

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: a , h , i Western blot of HDAC8 ( a ) or HIF1α ( h , i ) on lysates of rat SCs cultured in conditions mimicking the conversion into the repair phenotype under normoxia or hypoxia ( a , CoCl 2 for 16 h; i , hypoxic chamber for 5 h) or on lysates of contralateral (Co) and crushed (Cr) sciatic nerves of HDAC8 KO (H8KO) and control (Ctrl) mice at 1 dpl ( h ), after subcellular fractionation (Cyt = cytoplasmic, Nuc = nuclear) and quantification of the nuclear fraction normalized to Lamin A/C ( h ) or of the cytoplasmic fraction normalized to GAPDH ( h , i ) in cells where HDAC8 was downregulated by shRNA (H8sh) compared to control shRNA (Csh) ( i ), or in H8KO compared to Ctrl nerves ( h ). b – g , j HIF1α ( b – d , g ), c-Jun ( e ), phospho-c-Jun (p-c-Jun, f ) and phospho-JNK1/2 (p-JNK1/2, j ) Western blot on lysates of rat SCs cultured as above ( a ) in normoxia (N, b , c , g ) or hypoxia (H, 16 h ( b , d – f , j ) with CoCl 2 or ( c ) in hypoxia chamber), and ( g ) incubated with the proteasome inhibitor MG-132 or its vehicle for 4 h, and quantification normalized to GAPDH, β-actin or eEF1A1 in cells incubated with lentiviruses carrying H8sh ( b , c , g ) or a HIF1α shRNA (HFsh, d – f , j ) or Csh. k , l Phospho-c-Jun (p-c-Jun, k ) or c-Jun ( l ) Western blots on lysates of rat SCs cultured as above ( a ) in hypoxia and incubated with H8sh or Csh lentivirus, and with a JNK inhibitor (JNKi) or its vehicle (V), and quantification normalized to GAPDH. Dashed lines=samples run on the same gel but not on consecutive lanes. Three ( a ) or 6 ( d ) independent experiments, representative images are shown. h N = 4 (Ctrl) or 3 (H8KO) animals. N = 3 ( i , j , k , l ), 4 ( f ) or 6 ( b , c , e , g ) independent experiments. Paired ( b , c , e , g , i , j , k , l ) or unpaired ( f , h ) two-tailed (black asterisks) or one-tailed (gray asterisks, n.s.) Student’s t-tests, p values: *<0.05, **<0.01, n.s. = non-significant, values = mean, error bars = s.e.m. Source data are provided as a Source data file.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques: Western Blot, Cell Culture, Control, Fractionation, shRNA, Incubation, Two Tailed Test, One-tailed Test

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: a HDAC8 (H8) putative binding partners GO analysis after H8 immunoprecipitation (IP) on lysates of crushed and contralateral mouse sciatic nerves at 1 dpl and analysis of binding partners by mass spectrometry. IP Ctrl = negative control IP on same lysates as H8 IP. b , c TRAF7 Western blot after subcellular fractionation (Cyt = cytoplasmic, Nuc = nuclear) of b rat SCs cultured under normoxia or hypoxia (CoCl 2 for 16 h) in conditions mimicking the conversion into the repair phenotype or c crushed (Crush) and contralateral (Contra) mouse sciatic nerves at 1 dpl. GAPDH and Lamin A/C = markers of cytoplasmic and nuclear fractions, respectively. d IP H8 or IP Ctrl and TRAF7 or HDAC8 Western blot in lysates of rat SCs cultured as above ( b ) in normoxia. Same lysate separated into two equal fractions (for IP H8 and IP Ctrl). TRAF7, HDAC8 and GAPDH inputs (3% of IP lysate) are shown. e TRAF7 and GAPDH Western blots in lysates of rat SCs cultured as above ( b ) in normoxia and incubated with lentiviruses carrying a TRAF7 shRNA (T7sh) or a control shRNA (Csh), and quantification of short and long isoforms normalized to GAPDH. f TRAF7 Western blot in lysates of cells cultured as above ( b ) in normoxia and hypoxia and incubated with lentiviruses carrying an HDAC8 shRNA (H8sh) or Csh, and quantification normalized to GAPDH. g Co-immunofluorescence (z-series projections) of TRAF7 (red), HDAC8 (green) and Neurofilament (NF, magenta), and DAPI labeling (blue, nuclei) in cross-section of uninjured adult mouse sciatic nerves. h TRAF7 Western blot in lysates of HDAC8 KO (H8KO) and control (Ctrl) crushed (Cr) and contralateral (Co) mouse sciatic nerves at 1 dpl, and quantification normalized to GAPDH. Three independent IP ( a ), 3 animals ( c ), 5 independent experiments ( d ), sections of 3 WT mice analyzed ( g ), representative images are shown. N = 3 ( b ), 4 ( e ) or 6 ( f ) independent experiments. h N = 4 animals per group. Paired ( e , h /Ctrl-Co) or unpaired ( f , h /Ctrl-Cr) two-tailed (black asterisks) Student’s t-tests, p values: *<0.05, **<0.01, ***<0.001, n.s. = non-significant, values = mean, error bars = s.e.m. Source data are provided as a Source data file.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques: Binding Assay, Immunoprecipitation, Mass Spectrometry, Negative Control, Western Blot, Fractionation, Cell Culture, Incubation, shRNA, Control, Immunofluorescence, Labeling, Two Tailed Test

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: a , b HIF1α ( a ) or c-Jun ( b ) Western blot in lysates of rat SCs cultured in conditions mimicking the conversion into the repair phenotype in normoxia ( a ) or hypoxia ( b ) and incubated with lentiviruses carrying a TRAF7 shRNA (T7sh) or control shRNA (Csh), and quantification normalized to GAPDH. N = 9 ( a ) or 7 ( b ) independent experiments. c HIF1α Immunoprecipitation (IP) or control IP (Ctrl) and HIF1α Western blot in lysates of rat SCs cultured as above ( a ) and incubated with MG-132 for 8 h. Same lysate separated into two equal fractions (for HIF1α IP and Ctrl IP). GAPDH input=3% of IP lysate. Three independent experiments, representative images are shown. d IP HIF1α or Ctrl IP and TRAF7 Western blot in lysates of rat SCs cultured as above ( a ) and incubated for 8 h with MG-132. Three independent experiments, representative images are shown. Same lysate separated into two equal fractions, (for HIF1α IP and Ctrl IP). TRAF7 and GAPDH inputs=3% of IP lysate. e Denaturing HIF1α and Ctrl IP and ubiquitin (P4D1) Western blot in lysates of SCs transduced with T7sh or Csh lentivirus cultured as above ( a ) and incubated for 8 h with MG-132, and quantification of ubiquitinated HIF1α. N = 3 independent experiments. Each lysate separated into two equal fractions (for HIF1α IP and Ctrl IP). GAPDH input=3% of IP lysate. f TRAF7 Western blot on lysates of rat SCs transfected with a TRAF7- or Flag-expressing construct (Ctrl), and quantification normalized to GAPDH. N = 3 independent experiments. g – i HIF1α ( g ), c-Jun ( h ) or phospho-c-Jun (p-c-Jun, i ) Western blots on lysates of rat SCs cultured as above ( b ) and incubated with lentiviruses carrying an HDAC8 shRNA (H8sh) or Csh, and subsequently transfected with a TRAF7- or Flag-expressing construct (Ctrl), and quantification normalized to GAPDH. N = 3 independent experiments. Paired ( a , b , e , g , h , i /Ctrl) or unpaired ( f , i /Csh-TRAF7) two-tailed (black asterisks) or one-tailed (gray asterisks, n.s.) Student’s t-tests, p value: *<0.05, **<0.01, n.s. = non-significant, values = mean, error bars = s.e.m. Source data are provided as a Source data file.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques: Western Blot, Cell Culture, Incubation, shRNA, Control, Immunoprecipitation, Ubiquitin Proteomics, Transduction, Transfection, Expressing, Construct, Two Tailed Test, One-tailed Test

Journal: Nature Communications

Article Title: Hypoxia-induced conversion of sensory Schwann cells into repair cells is regulated by HDAC8

doi: 10.1038/s41467-025-55835-9

Figure Lengend Snippet: In WT SCs, HDAC8 interacts with and stabilizes TRAF7 short isoform, at the expense of TRAF7 long isoform; in turn, TRAF7 short isoform interacts with HIF1α and ubiquitinates it to target it to the proteasome for degradation. In the absence of HDAC8, TRAF7 short isoform is degraded, which leads to increased HIF1α levels. In parallel, TRAF7 long isoform is stabilized. Both HIF1α and TRAF7 long isoform increase the levels of phosphorylated JNK, HIF1α and phosphorylated JNK translocate to the nucleus, leading to increased phosphorylated c-Jun and total c-Jun levels. This mechanism promotes the conversion of sensory SCs into the repair phenotype and accelerates the regrowth of sensory axons and recovery of the sensory function.

Article Snippet: The day after, 30 μl of agarose beads were added and samples were further rotated at 4 °C for 2 h. Two to six micrograms of the following antibodies were used per nerve or per 1 × 10 7 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1).

Techniques:

Journal: Heliyon

Article Title: ZNF521 promotes acute myeloid leukemogenesis by suppressing the expression and acetylation of SMC3

doi: 10.1016/j.heliyon.2024.e37528

Figure Lengend Snippet: ZNF521 promotes HDAC8 expression, but suppresses SMC3 expression and acetylation. A-D:Quantitative RT-PCR analysis of CDK2 (A), CDK6 (B), HDAC8 (C), and SMC3 (D) were performed after THP-1 cells were transduced with shZNF521 for 72 h. E: western blotting analysis of SMC3 expression and acetylation and HDAC8 expression after THP-1 cells were transduced with shZNF521 for 72 h. Data are presented as the mean ± SD of three independent experiments. *: p < 0.05, **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The membrane was blocked with 5 % skimmed milk in TBST buffer at RT for 3 h, and then incubated overnight at 4 °C with primary antibodies against ZNF521 (cat no. TA319089, Origene, USA), HDAC8 (Cat No. TA809689, Origene, USA), SMC3 (Cat No. 14185-1-AP, Proteintech, China), ac-SMC3 (Cat No. MABE1073, Sigma, USA), and GAPDH (Cat No. 60004-1-Ig, Proteintech, China).

Techniques: Expressing, Quantitative RT-PCR, Transduction, Western Blot

Journal: Heliyon

Article Title: ZNF521 promotes acute myeloid leukemogenesis by suppressing the expression and acetylation of SMC3

doi: 10.1016/j.heliyon.2024.e37528

Figure Lengend Snippet: ZNF521 interacts with HDAC8. A: The interaction of ZNF521 and HDAC8 by immunoprecipitation in THP-1 cells. B: immunofluorescent analysis of ZNF521 and HDAC8 in THP-1 cells. C: the interaction of ZNF521 (blue) and HDAC8 (red) predicted by AlphaFold 3.

Article Snippet: The membrane was blocked with 5 % skimmed milk in TBST buffer at RT for 3 h, and then incubated overnight at 4 °C with primary antibodies against ZNF521 (cat no. TA319089, Origene, USA), HDAC8 (Cat No. TA809689, Origene, USA), SMC3 (Cat No. 14185-1-AP, Proteintech, China), ac-SMC3 (Cat No. MABE1073, Sigma, USA), and GAPDH (Cat No. 60004-1-Ig, Proteintech, China).

Techniques: Immunoprecipitation

Journal: Heliyon

Article Title: ZNF521 promotes acute myeloid leukemogenesis by suppressing the expression and acetylation of SMC3

doi: 10.1016/j.heliyon.2024.e37528

Figure Lengend Snippet: The interaction of ZNF521 and HDAC8 promotes proliferation and inhibits apoptosis by suppressing SMC3. A: SMC3 expression and acetylation is inhibited by ZNF521 and HDAC8. Upper panel: the WB and down panel: graphic analysis of WB. B–E: proliferation (B and C) and apoptosis (D–E) were analyzed after HL-60 cells were transduced with ZNF521 overexpression plasmid, and treated with siHDAC8 or HDAC8 inhibitor, and siSMC3 for 72 h. Data are presented as the mean ± SD of three independent experiments. Ns: not significant, **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The membrane was blocked with 5 % skimmed milk in TBST buffer at RT for 3 h, and then incubated overnight at 4 °C with primary antibodies against ZNF521 (cat no. TA319089, Origene, USA), HDAC8 (Cat No. TA809689, Origene, USA), SMC3 (Cat No. 14185-1-AP, Proteintech, China), ac-SMC3 (Cat No. MABE1073, Sigma, USA), and GAPDH (Cat No. 60004-1-Ig, Proteintech, China).

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation

Journal: Heliyon

Article Title: ZNF521 promotes acute myeloid leukemogenesis by suppressing the expression and acetylation of SMC3

doi: 10.1016/j.heliyon.2024.e37528

Figure Lengend Snippet: The model of ZNF521interacting with HDAC8 to suppress the expression and acetylation of SMC3.

Article Snippet: The membrane was blocked with 5 % skimmed milk in TBST buffer at RT for 3 h, and then incubated overnight at 4 °C with primary antibodies against ZNF521 (cat no. TA319089, Origene, USA), HDAC8 (Cat No. TA809689, Origene, USA), SMC3 (Cat No. 14185-1-AP, Proteintech, China), ac-SMC3 (Cat No. MABE1073, Sigma, USA), and GAPDH (Cat No. 60004-1-Ig, Proteintech, China).

Techniques: Expressing

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 HDAC4 (His6-HDAC4) (31364), HDAC7 (His6-HDAC7) (31535) and HDAC8 (His6-HDAC8) (Ag11692) proteins 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: 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 HDAC4 (His6-HDAC4) (31364), HDAC7 (His6-HDAC7) (31535) and HDAC8 (His6-HDAC8) (Ag11692) proteins 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 HDAC4 (His6-HDAC4) (31364), HDAC7 (His6-HDAC7) (31535) and HDAC8 (His6-HDAC8) (Ag11692) proteins were purchased from Proteintech (China).

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