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: Molecular and Cellular Biology

Article Title: Histone Deacetylase 8 Safeguards the Human Ever-Shorter Telomeres 1B (hEST1B) Protein from Ubiquitin-Mediated Degradation

doi: 10.1128/mcb.01971-05

Figure Lengend Snippet: FIG. 1. hEST1B interacts with HDAC8 in a phosphorylation-dependent manner. (A) PKA-mediated HDAC8 phosphorylation increases its association with hEST1B. HA-EST1B and Flag-HDAC8, either wild type or mutant, were coexpressed in HeLa cells in the absence or presence of forskolin. Following immunoprecipitation with an anti-HA antibody, the samples were resolved by SDS-PAGE and the presence of coimmu- noprecipitated HDAC8 was detected by immunoblotting with an anti-Flag antibody. (B) Foskolin induces the phosphorylation of wild-type but not S39A mutant HDAC8. Flag-tagged wild-type or S39A mutant HDAC8 was transfected into HeLa cells in the presence or absence of forskolin. Following immunoprecipitation with anti-Flag antibodies, the samples were resolved by SDS-PAGE and phosphorylated HDAC8 was detected with an anti-phospho-serine antibody. (C) Phosphorylated HDAC8 preferentially interacts with hEST1B, but not hEST1A. Plasmids that express the indicated EST1 were transfected into cells, together with a plasmid that expresses HDAC8 in the presence of forskolin. (D) Endogenous hEST1B and HDAC8 interact. HeLa cells were treated with either forskolin or H-89 as indicated, and lysates were immunoprecipitated with anti-hEST1B antibodies. The immunoprecipitates were washed, and the proteins were separated by SDS-PAGE. Coimmunoprecipitated HDAC8 was detected by immunoblotting using TrueBlot to reduce the background due to the immunoglobulin G heavy chain. All protein concentrations were adjusted according to the expression levels of HA-EST1B. For example, in panel A, the reaction displayed in lane 2 contains three times more lysate than that in lane 3. , absence of; , presence of; IP, immunoprecipitate; IB, immunoblot; PIS, preimmune serum.

Article Snippet: A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunoprecipitates.

Techniques: Phospho-proteomics, Mutagenesis, Immunoprecipitation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Molecular and Cellular Biology

Article Title: Histone Deacetylase 8 Safeguards the Human Ever-Shorter Telomeres 1B (hEST1B) Protein from Ubiquitin-Mediated Degradation

doi: 10.1128/mcb.01971-05

Figure Lengend Snippet: FIG. 2. HDAC8 phosphorylation protects hEST1B protein from ubiquitin-mediated degradation. (A) HA-EST1B and Flag-HDAC8 were expressed in HeLa cells in the absence or presence of forskolin and analyzed by Western blotting. Western blotting with anti--actin (right panel) was used as a control. (B) Western blot analyses of extracts derived from HeLa cells or a HeLa cell line that overexpresses HDAC8 (MC5HD8). (C) Western blot analyses from extracts of adenovirus-infected cells to determine the effects of overexpression of HDAC8 on hEST1B. (D) Left panel, luciferase reporter assays showing that overexpression of HDAC8 does not alter the transcription of the cytomegalovirus promoter (pCMV-Luc). Transfections were normalized to equal amounts of DNA with parental expression vector. The results are the means standard deviations from three separate transfections. Right panel, semiquantitative RT-PCR was performed to analyze expression of the hEST1B gene. PCR products of cDNA samples are shown. (E) MG132 blocks hEST1B degradation. Cells were treated with 10 M MG132 for 18 h before harvesting. hEST1B protein was detected by an anti-HA antibody. (F) HDAC8 phosphorylation protects hEST1B from degradation. Cells were transfected with His-ubiquitin, together with wild-type or mutant HDAC8, in the presence of HA-EST1B as indicated. (G) Inhibition of hEST1B ubiquitination by phosphorylated HDAC8. The level of hEST1B ubiquitination was determined by using an anti-ubiquitin antibody in the presence of His-ubiquitin after treatment with MG132. The amount of protein used in this experiment was adjusted according to the expression level shown in panel A. Ub, ubiquitin; , absence of; , presence of; IP, immunoprecipitate; IB, immunoblot.

Article Snippet: A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunoprecipitates.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Western Blot, Control, Derivative Assay, Infection, Over Expression, Luciferase, Transfection, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Histone Deacetylase 8 Safeguards the Human Ever-Shorter Telomeres 1B (hEST1B) Protein from Ubiquitin-Mediated Degradation

doi: 10.1128/mcb.01971-05

Figure Lengend Snippet: FIG. 3. hEST1B ubiquitination is not affected by its acetylation. (A) HA-EST1B and Flag-tagged wild-type, S39A mutant, or H143A mutant HDAC8 were coexpressed in HeLa cells. After the lysate was resolved by SDS-PAGE, the hEST1B protein level was determined by immuno- blotting using an anti-HA antibody. (B) hEST1B is acetylated in vivo. Proteins expressing HA-EST1B were immunoprecipitated using an anti-HA antibody. For negative controls, preimmune serum (PIS) and anti-Flag antibodies were used. Acetylated hEST1B proteins were detected by immunoblotting with an anti-acetyl lysine (anti-Ac-K) antibody. To confirm equal amounts of HA-EST1B in the lysates, a small portion was removed before antibody incubation and resolved by SDS-PAGE. (C) hEST1B ubiquitination is not affected by TSA treatment. Cells were treated with 400 ng/ml TSA for 18 h and MG132 for 5 h before harvesting. Ub, ubiquitin; , absence of; , presence of; IP, immunoprecipitate; IB, immunoblot.

Article Snippet: A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunoprecipitates.

Techniques: Ubiquitin Proteomics, Mutagenesis, SDS Page, In Vivo, Expressing, Immunoprecipitation, Western Blot, Incubation

Journal: Molecular and Cellular Biology

Article Title: Histone Deacetylase 8 Safeguards the Human Ever-Shorter Telomeres 1B (hEST1B) Protein from Ubiquitin-Mediated Degradation

doi: 10.1128/mcb.01971-05

Figure Lengend Snippet: FIG. 4. HDAC8 recruits Hsp70 and CHIP to hEST1B in a phosphorylation-dependent manner. (A) HeLa cells were transfected with either wild-type or S39A HDAC8, and lysates were immunoprecipitated with an anti-Flag antibody. The presence of Hsp proteins was determined using anti-Hsp70 or anti-Hsp90 antibodies. (B) HDAC8-Hsp protein complexes were immunoprecipitated as described above. The amount of protein used was adjusted according to the expression level shown in Fig. 2A. (C) hEST1B is a CHIP ubiquitin ligase-interacting protein. HA-EST1B and Myc-CHIP were expressed in HeLa cells and harvested after 48 h for analysis. (D) CHIP recruitment to hEST1B is dependent on HDAC8 phosphorylation. Myc-CHIP constructs were transfected with HA-EST1B and Flag-HDAC8 in the presence or absence of forskolin. The ability of CHIP to interact with hEST1B was determined with an anti-Myc antibody. , absence of; , presence of; IP, immunoprecipitate; IB, immunoblot.

Article Snippet: A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunoprecipitates.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Expressing, Ubiquitin Proteomics, Construct, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Histone Deacetylase 8 Safeguards the Human Ever-Shorter Telomeres 1B (hEST1B) Protein from Ubiquitin-Mediated Degradation

doi: 10.1128/mcb.01971-05

Figure Lengend Snippet: FIG. 5. CHIP is an E3 ubiquitin ligase specific for hEST1B, and HDAC8 phosphorylation regulates CHIP-mediated degradation of hEST1B. (A) Ubiquitinated hEST1B proteins were visualized with an anti-ubiquitin antibody in the lysates from cells expressing HA-EST1B and various Myc-CHIP constructs. (B) An in vitro ubiquitination assay was performed using bacterially expressed proteins as indicated. After the reaction, Ni-nitrilotriacetic acid agarose-bound proteins were washed extensively and resolved by SDS-PAGE. Ubiquitin conjugation on hEST1B proteins was detected with an anti-Flag antibody. (C) An in vitro ubiquitination assay was performed using the bacterially expressed CHIP deletion mutant. (D) cDNAs expressing HA-EST1B, Flag-HDAC8, and Myc-CHIP were transfected into HeLa cells, and immunoblots were performed as indicated. Ub, ubiquitin; , absence of; , presence of; IP, immunoprecipitate; IB, immunoblot.

Article Snippet: A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunoprecipitates.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Expressing, Construct, In Vitro, SDS Page, Conjugation Assay, Mutagenesis, Transfection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Histone Deacetylase 8 Safeguards the Human Ever-Shorter Telomeres 1B (hEST1B) Protein from Ubiquitin-Mediated Degradation

doi: 10.1128/mcb.01971-05

Figure Lengend Snippet: FIG. 6. Silencing of HDAC8 results in hEST1B degradation and the down-regulation of total telomerase activity. A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunopre- cipitates. Telomerase activity was visualized by autoradiography following resolution on nondenaturing gels. As a negative control for hEST1B- specific telomerase interaction, immunoprecipitates with preimmune serum (PIS) were used (lane 4). A positive control for telomerase activity is shown in lane 1. IC, internal control. (C) Total telomerase activity was measured using a different amount of lysates. For a negative control, heat-inactivated lysate was prepared from control siRNA-transfected cells (lane 1). , absence of; , presence of; IP, immunoprecipitate; IB, immunoblot.

Article Snippet: A TRAP assay was performed to detect hEST1B-associated telomerase and total telomerase activity in HDAC8 knockdown cells. (A) Two different pools of HDAC8 siRNA oligonucleotides purchased from Santa Cruz Biotechnology (sc-35548, lane 2) and Dharmacon (M-003500-02, lane 4) were used to knock down HDAC8 expression, and the effects on hEST1B protein levels were examined. (B) The TRAP assay was performed using 2 l of immunoprecipitates.

Techniques: Activity Assay, TRAP Assay, Knockdown, Expressing, Autoradiography, Negative Control, Positive Control, Control, Transfection, Western Blot

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: