Journal: Nature Communications

Article Title: Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

doi: 10.1038/ncomms3532

Figure Lengend Snippet: ( a , b ) Immunoblot for endogenous HDAC6 ( a ) in newborn (P0) and adult cerebral cortex and ( b ) in primary cerebrocortical neurons at div2 and 3. Each lane respectively contained 50/10 μg total protein. ( c ) Mutual co-immunoprecipitation of endogenous SEPT7 and HDAC6. Lysates from div2 cerebrocortical neurons were incubated with protein A beads coated with nonimmune IgG, anti-SEPT7 antibody or anti-HDAC6 antibody, and these proteins were detected by immunoblot in a reciprocal manner. ( d ) Co-expression and mutual co-immunoprecipitation of GFP-SEPT7 and Flag-HDAC6 in heterologous cells. (Left) Anti-Flag antibody detected Flag-HDAC6 (and gave nonspecific faint bands in the 1st and 3rd lanes) and anti-GFP antibody detected GFP and GFP-SEPT7. (Right) When Flag- HDAC6 was captured on anti-Flag beads, GFP-SEPT7 was co-immunoprecipitated, but GFP was not (compare the 2nd and 4th lanes. *, IgG light chain). ( e ) Direct binding between the purified, recombinant septin complex and HDAC6 in vitro . Immobilized His-tagged SEPT7/6/2 captured GST-HDAC6 but not GST alone. (Coomassie blue staining). ( f ) Representative results of in situ proximity ligation assay for endogenous SEPT7 and HDAC6 in div2 cerebrocortical neurons. Fluorescent puncta were generated in the somata and neurites only when anti-SEPT7 antibodies and anti-HDAC6 antibodies were present in close proximity. Scale bars, 25 μm. ( g ) Immunoblot showing that SEPT7 depletion did not affect the amount of HDAC6 in div2 cerebrocortical neurons. (Triplicated experiment. NS, P >0.05 by t -test). Error bars denote s.e.m. ( h ) SEPT7 depletion via RNAi (#1, S7KD) did not alter the deacetylating activity of HDAC6. Cell lysates as in ( b ) were subjected to an in vitro assay with a fluorogenic substrate. (Triplicated experiment. NS, P >0.05 by t -test). Error bars denote s.e.m. ( i ) SEPT7 depletion via RNAi (#1, S7KD) caused dissociation of HDAC6 and acetylated α-tubulin. (Left) Cell lysates as in ( b , h ) were immunoblotted for endogenous HDAC6, acetylated α-tubulin and total α-tubulin. Hyperacetylation of α-tubulin in SEPT7-depleted neurons was recapitulated ( cf. ). Each lane contained 50 μg total protein. (Right) Although SEPT7 depletion increased acetylated α-tubulin, its association with HDAC6 was paradoxically reduced. (Triplicated experiments. * P <0.05, ** P <0.01 by t -test). Error bars denote s.e.m.

Article Snippet: Sf9-expressed His 6 -tagged SEPT7/6/2 (ref. ) was immobilized on Ni-NTA beads (Qiagen) and incubated with Sf9-expressed GST-tagged human HDAC6 (SignalChem) or GST alone in PBS for 6 h at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Incubation, Expressing, Binding Assay, Purification, Recombinant, In Vitro, Staining, In Situ, Proximity Ligation Assay, Generated, Activity Assay

Journal: Nature Communications

Article Title: Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

doi: 10.1038/ncomms3532

Figure Lengend Snippet: ( a ) (Top panels) Representative images of GFP-expressing cerebrocortical neurons at div2 treated with vehicle alone (0.1% DMSO) or with 10 μM tubacin in vehicle. HDAC6 inhibition by tubacin inhibited the growth of both dendrites and axons. (Bottom panels) SEPT7 depletion via RNAi (#1) combined with tubacin treatment did not exhibit an obvious additive effect on neurite morphology. Scale bar, 50 μm. ( b ) Scattergram of total dendrite length and total axon length of pyramidal-like cerebrocortical neurons treated with tubacin and SEPT7-depletion in the four combinations shown in ( a ). The growth of both dendrites and axons was markedly inhibited by tubacin treatment (black closed circles) in comparison with the control (black open circles), which was not enhanced by additional depletion of SEPT7 (red closed circles). ( c ) Statistical analysis of the results shown in ( b ) with plots of the mean values, showing that tubacin treatment significantly and proportionally shortened dendrites and axons. The neurite-shortening effect of tubacin was more potent than, and not enhanced by, SEPT7 depletion. The grey bars represent average±2 s.e.m. of the control samples. ( n =45 × 4. *** P <0.001 by one-way ANOVA with post hoc Tukey). Error bars denote s.e.m. ( d and e ) Tip numbers of axons and dendrites in the above samples were significantly reduced by tubacin alone, by SEPT7 depletion alone, or by the combination of the two. ( n =45 × 4. *** P <0.001 by one-way ANOVA with post hoc Tukey). Error bars denote s.e.m.

Article Snippet: Sf9-expressed His 6 -tagged SEPT7/6/2 (ref. ) was immobilized on Ni-NTA beads (Qiagen) and incubated with Sf9-expressed GST-tagged human HDAC6 (SignalChem) or GST alone in PBS for 6 h at 4 °C.

Techniques: Expressing, Inhibition, Comparison, Control

Journal: Nature Communications

Article Title: Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

doi: 10.1038/ncomms3532

Figure Lengend Snippet: HDAC6 is the major microtubule deacetylase, which promotes microtubule remodelling by counteracting acetyl transferases that stabilize microtubules . This study demonstrated that the direct interaction with the septin complex facilitates the access of HDAC6 to acetylated α-tubulin and/or stabilizes the enzyme-substrate interaction without altering the deacetylation activity of HDAC6. An open question is whether the putative tripartite interaction of the septin complex/HDAC6/acetylated α-tubulin (a subset of the signals in the PLA assay shown in should represent this) occurs on α/β-tubulin heterodimers, protofilaments and/or microtubules. It is also worth testing whether an actomyosin-dependent mechanism, including another HDAC6 substrate cortactin , could contribute to the stagnant neurite growth after septin depletion. Independent of the specific underlying mechanism, the novel molecular network identified in this study has shed new light on the common machinery for the growth of axons and dendrites in vivo and in vitro .

Article Snippet: Sf9-expressed His 6 -tagged SEPT7/6/2 (ref. ) was immobilized on Ni-NTA beads (Qiagen) and incubated with Sf9-expressed GST-tagged human HDAC6 (SignalChem) or GST alone in PBS for 6 h at 4 °C.

Techniques: Histone Deacetylase Assay, Activity Assay, In Vivo, In Vitro

Journal: Cancer Immunology, Immunotherapy

Article Title: HDAC6 inhibitor ACY-1215 enhances STAT1 acetylation to block PD-L1 for colorectal cancer immunotherapy

doi: 10.1007/s00262-023-03624-y

Figure Lengend Snippet: The HDAC6 inhibitor (ACY-1215) plays an additive antitumor effect with anti-PD1. A The antitumor effects of ACY-1215 and the immune checkpoint inhibitors (anti-PD1) in vivo were investigated in a mouse transplanted tumor model. Please refer to “Materials and Methods” for specific construction methods of animal models. B 21st days after MC38 cells injection, the C57BL/6 mice were sacrificed to observe tumor mass. C Tumor growth curve of each group. The tumor volume was measured every two days from day 9 to 21 after MC38 cell inoculation. D Photomicrographs illustrating hematoxylin-eosin staining in the heart, liver, spleen, lung, and kidney of the tumorous mice. Scale bar: 100 μm. E The expressions of CD8a, TBX21 and Granzyme B in tumor tissues of tumor-bearing mice were assayed by immunohistochemistry and quantitatively analyzed. Scale bar: 50 μm

Article Snippet: Appropriate amount of acetylated STAT1 was added to human recombinant HDAC6 (Active Motif, Cat#31,543) and HDAC6 deacetylation buffer II (50 mM Tris-HCl pH8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mg/mL BSA).

Techniques: In Vivo, Injection, Staining, Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy

Article Title: HDAC6 inhibitor ACY-1215 enhances STAT1 acetylation to block PD-L1 for colorectal cancer immunotherapy

doi: 10.1007/s00262-023-03624-y

Figure Lengend Snippet: ACY-1215 down-regulates PD-L1 by promoting STAT1 acetylation. HCT116 cells were pretreated with 20 ng/mL IFN-γ, and then treated with ACY-1215 (0, 2.5, 5.0 µM) for 48 h to extract the total protein of the cells for Western blot experiments, or treated with ACY-1215 (0, 5.0 µM) for 24 h in immunofluorescence experiments. A , B The expression of phosphorylated STAT1 (p-STAT1) and NF-κB p65 in whole cells were detected by Western Blot ( A ); the expression of STAT1 proteins in cytoplasm and nucleus was detected by Western blot ( B ). GAPDH is the cytoplasmic reference protein and H3 is the nuclear reference protein. C The effect of ACY-1215 on STAT1 entry into the nucleus was detected by immunofluorescence. STAT1 was the green fluorescence, the nucleus was stained by DAPI, “Merge” represents the signal superposition diagram of STAT1 and DAPI. Scale bar: 50 μm. D The effects of ACY-1215 on IFN-γ-induced STAT1 phosphorylation and acetylation were determined by immunoprecipitation. E The expression of NF-κB p65 in cytoplasm and nucleus was detected by Western blot. GAPDH is the cytoplasmic reference protein and H3 is the nuclear reference protein. F The effect of ACY-1215 on NF-κB p65 entry into the nucleus was detected by immunofluorescence. NF-κB p65 was the red fluorescence, the nucleus was stained by DAPI, “Merge” represents the signal superposition diagram of NF-κB p65 and DAPI. Scale bar: 50 μm. G and H Once adhering to the plates, HCT116 cells were co-transfected with luciferase reporter vectors constructed with the PD-L1 promoters, pRL-TK Renilla luciferase vector, STAT1 plasmid vector ( G ) or NF-κB p65 plasmid vector ( H ). After transfection for 24 h, the transfected HCT116 cells were pretreated with 20 ng/mL IFN-γ, and then treated with ACY-1215 for 24 h. The cell lysates were collected and the activity of luciferase was detected. I The endogenous interaction between STAT1 and HDAC6 in HCT116 cells was analyzed by co-immunoprecipitation. J The co-localization of STAT1 and HDAC6 in HCT116 cells was detected by immunofluorescence. Scale bar: 50 μm. K The fluorescence signal distribution of STAT1 and HDAC6 was measured by ImageJ software. Three independent repeated experiments were performed. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, in two-tailed unpaired t test

Article Snippet: Appropriate amount of acetylated STAT1 was added to human recombinant HDAC6 (Active Motif, Cat#31,543) and HDAC6 deacetylation buffer II (50 mM Tris-HCl pH8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mg/mL BSA).

Techniques: Western Blot, Immunofluorescence, Expressing, Fluorescence, Staining, Phospho-proteomics, Immunoprecipitation, Transfection, Luciferase, Construct, Plasmid Preparation, Activity Assay, Software, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy

Article Title: HDAC6 inhibitor ACY-1215 enhances STAT1 acetylation to block PD-L1 for colorectal cancer immunotherapy

doi: 10.1007/s00262-023-03624-y

Figure Lengend Snippet: The mechanisms by which HDAC6 inhibitor (ACY-1215) down-regulates PD-L1 and promotes T cell activation. (Upper) In the absence of HDAC6 inhibitors, the activity of HDAC6 in tumor cells is high. HDAC6 interacts with STAT1. HDAC6 deacetylates STAT1, which keeps low STAT1 acetylation. When the NF-κB and STAT1 signaling pathways are activated, NF-κB p65 and STAT1 enter the nucleus and promote the activation of downstream target genes, including PD-L1. The highly expressed PD-L1 (ligand) binds to the PD-1 (receptor) on the surface of T cells, inhibits the activity of T cells, enabling immune escape of tumor cells. (Bottom) When treating tumor cells with the HDAC6 inhibitors (ACY-1215), ACY-1215 inhibits the entry of STAT1 and NF-κB p65 into the nucleus to exert transcriptional regulation on PD-L1 by promoting the acetylation of STAT1, leading to downregulation of PD-L1 expression and thus promoting the activation of T cells

Article Snippet: Appropriate amount of acetylated STAT1 was added to human recombinant HDAC6 (Active Motif, Cat#31,543) and HDAC6 deacetylation buffer II (50 mM Tris-HCl pH8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mg/mL BSA).

Techniques: Activation Assay, Activity Assay, Protein-Protein interactions, Expressing