Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: List of primary antibodies used in this study

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: Knockout of HDAC6 alleviates brain damage following ICH. A–B HDAC6−/− mice had defects in HDAC6 gene amplification and HDAC6 protein expression compared with WT mice. At day 3 following ICH, C–D Nissl staining and analysis were employed to evaluate hematoma volume (n = 6 mice/group), E–F mNSS score and corner turn test were applied to detect neurological dysfunction (n = 10 mice/group), and G brain water content analysis was used to assess cerebral edema (n = 6 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques: Knock-Out, Amplification, Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: Knockout of HDAC6 alleviates oxidative stress following ICH. A In vivo experimental design for results presented in Fig. 2B–C. B–C Malondialdehyde (MDA) level and NADPH/NADP+ ration in HDAC6−/− and control WT mice at day 3 following ICH (n = 6/group). D The effect of HDAC6 siRNA was verified by WB assay (n = 3/group). E Representative fluorescence images showing DCFH-DA labeled ROS levels at 24 h following hemin exposure. F Quantitation of ROS content in the group of E (n = 6/group). G Representative images of neuron death shown by double TUNEL and NeuN staining in the peri-hematoma zone of HDAC6−/− and control WT mice at day 3 following ICH (neuron: green and TUNEL: red) and magnified images of NeuN+/TUNEL+ staining were indicated with white dashed line squares. Scale bars: 50 μm. H–L Quantitative analysis of H total NeuN+ cells, I total TUNEL+ cells, J double NeuN+/TUNEL+ cells, K the ration of NeuN+/TUNEL+ cells among total NeuN+ cells, and L the ratio of NeuN+/TUNEL+ cells among total TUNEL+ cells in all groups of G (n = 6/group). M Representative western blot (WB) images and quantitative analysis of cleaved caspase-3 and Bax proteins at day 3 following ICH in vivo (n = 3/group). N Representative WB images and quantitative analysis of cleaved caspase-3 and Bax protein at 24 h following hemin exposure (n = 3/group). *P < 0.05, ***P < 0.001

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques: Knock-Out, In Vivo, Control, Fluorescence, Labeling, Quantitation Assay, TUNEL Assay, Staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: Interaction between HDAC6 and MDH1. A 293T cells were single-transfected with Flag-HDAC6 plasmid or con-transfected with Myc-MDH1 plasmid. A total of 1 mg cell samples were immunoprecipitated by 1 μg of anti-Flag or anti-Myc antibody 24 h after transfection. Immunoprecipitants were then subjected to WB analysis with the indicated antibodies. B Brain samples of WT mice were subjected to IP assays with control anti-IgG or anti-HDAC6 or anti-MDH1 antibody, followed by WB analysis with the indicated antibodies. C Typical confocal images for HDAC6 (red) and MDH1 (green) in 293T and HT22 cells that co-transfected with Flag-HDAC6 and Myc-MDH1 plasmids. D Cell lysates of 293T cells transfected with Myc-MDH1 plasmid alone or combined with Flag-HDAC6 plasmid were precipitated by anti-acetyl-lysine antibody. WB analysis of acetylated MDH1 level was then conducted. E 293T cells transfected with Flag-HDAC6 were treated with 10 μM TubA. Cells were further subjected to IP assay to determine ac-MDH1 level. F Immunoprecipitation of brain tissue lysates of WT and HDAC6−/− mice using anti-acetyl-lysine antibody, followed by WB analysis of ac-MDH1 level

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: HDAC6 negatively regulates MDH1 acetylation in response to ICH. A–C IP and WB analysis of the ac-MDH1 in HT22 cells at 24 h following treated with A Hemin, B Hemoglobin, and C Thrombin. D IP assay performed to determine ac-MDH1 level in the indicated group (n = 3/group). E IP assay determined to examine acetylation level of MDH1 in the indicated group (n = 3/group). F IP and WB analysis of the acetylation level of MDH1 in HDAC6−/− and WT mice at day 3 after subjection to ICH surgery (n = 3/group). G coIP assay of the interaction between HDAC6 and MDH1 at day 3 after ICH surgery or under 30 mg/kg TubA treatment in WT mice. *P < 0.05, ***P < 0.001

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques: Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: HDAC6 deacetylates lysine residues of MDH1 at position 121 and 298. A 293T cells were transfected with wild-type and mutated plasmids of Myc-MDH1 (K118R, K121R, K298R, 3KR) for 24 h, and then subjected to IP and immunoblotting assay with the indicated antibodies. B 293T cells were co-transfected with Flag-HDAC6 and Myc-MDH1 (WT, K118R, K121R, K298R, 3KR) plasmids for 24 h, and then subjected to IP and immunoblotting assay with the indicated antibodies. C 293T cells were transfected with Myc-MDH1 (WT, K121R, K121Q, K298R, K298Q) plasmids for 24 h, and then subjected to IP and immunoblotting assay with the indicated antibodies. D 293T or E HT22 cells were transfected with Myc-MDH1 plasmids (WT, 2KR, 2KQ) for 24 h and then subjected to IP and immunoblotting assay with the indicated antibodies

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques: Transfection, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage

doi: 10.1007/s00018-022-04341-y

Figure Lengend Snippet: Protective effect of HDAC6 inhibition depends upon acetylation level of MDH1. A Experimental design for results presented in Fig. 7B–H. B–C Results of green-fluorescent protein (GFP)-MDH1 lentivirus in the brain were identified by B autofluorescence without primary antibody staining and C WB assay (n = 3/group). At day 3 following ICH, D–E brain injury was assayed by Nissl staining (n = 6/group), F cerebral edema was determined by the brain water content (n = 6/group), and G–H neurological function was assayed by mNSS score and corner-turning test (n = 10/group). I Model illustration of mechanism by which HDAC6/MDH1 signaling mediates oxidative stress-induced neuron apoptosis following ICH. Upon ICH injury, the interaction between HDAC6 and MDH1 was enhanced, which promoted HDAC6 mediated-MDH1 deacetylation at K121 and K298, thereby inhibiting NADP+ shift to NADPH; consequently, ROS was overproduced, which contributed to neuron apoptosis. However, administration of TubA (a specific HDAC6 inhibitor) alleviated the oxidative stress response by disrupting HDAC6 and MDH1 association, thus recovered neuron from apoptosis. **P < 0.01, ***P < 0.001

Article Snippet: The HDAC6 small interfering RNA (siRNA) used in this study was purchased from GenePharma (Suzhou, China) and transfected into cells with lipofectamine 2000 reagent according to the manufacturer’s protocols.

Techniques: Inhibition, Staining