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

Journal: The FASEB Journal

Article Title: Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex

doi: 10.1096/fj.201902010RR

Figure Lengend Snippet: Effects of NS5806 on cloned Kv4.3/KChIP2/DPP6‐L channels in HEK293 cells. A, Representative current traces elicited by the depolarizing voltage steps from −40 to +40 mV for 2 seconds from a holding potential of −80 mV at different transfection ratios of Kv4.3: KChIP2: DPP6‐L (Left). The superimposed current traces at +40 mV in the absence and presence of NS5806 are shown on the right. B, Effect of 10 μM NS5806 on the peak current of the Kv4.3/KChIP2/DPP6‐L currents produced by different subunit transfection ratios, measured at +40 mV. C, The time constant of inactivation ( τ ) of Kv4.3/KChIP2/DPP6‐L currents produced by different subunit transfection ratios (n = 22). D, I–V relationships of Kv4.3/KChIP2/DPP6‐L peak current density at plasmid ratio 1:1:1 before and after 10 μM NS5806 (n = 18, * P < .05, ** P < .01 vs control). E, Steady‐state inactivation curves for Kv4.3/KChIP2/DPP6‐L channel complex at plasmid ratio 1:1:1 before and after 10 μM NS5806 (n = 18). F, Recovery from inactivation curves for Kv4.3/KChIP2/DPP6‐L channel complex at plasmid ratio1:1:1 before and after 10 μM NS5806 (n = 18)

Article Snippet: The small interfering RNA (siRNA) duplex against the human DPP6 gene (NM_130797, 5′‐GGTCCATCATCGGCTCTTT‐3′) was designed and constructed by Ribobio (Guangdong, China). hiPSC‐CMs were transfected with siRNA using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instruction at a final siRNA concentration of 100 nM.

Techniques: Clone Assay, Transfection, Produced, Plasmid Preparation, Control

Journal: The FASEB Journal

Article Title: Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex

doi: 10.1096/fj.201902010RR

Figure Lengend Snippet: DPP6 expression in mouse, canine ventricular tissue, and hiPSC‐CMs. A 1 , Representative immunoblots of DPP6 protein expression in mouse left ventricle, canine left ventricle, and hiPSC‐CMs. A 2 , Test for antibody specificity by preincubation with the antigen peptide. B 1 , Efficiency of siRNA‐mediated DPP6 knockdown (SiDPP6) in hiPSC‐CMs quantified by RT‐PCR and compared to non‐targeting control siRNA (SiNC) (N = 3). B 2 , Representative immunoblots of DPP6 in hiPSC‐CMs treated with siRNA targeting DPP6 (N = 3). B 3 , Time constants ( τ ) of the current inactivation in hiPSC‐CMs treated with siRNA targeting DPP6 and a non‐targeting control. Data obtained by fitting a single exponential equation (n = 9, * P < .05, ** P < .01 vs SiNC). C 1 , Representative I to current traces recorded from the hiPSC‐CMs treated with SiNC or SiDPP6. C 2 , Effect of siRNA knockdown of DPP6 on the I–V relationships of I to and on the efficacy of NS5806‐mediated current inhibition in hiPSC‐CMs (left). Summary of the inhibitory effect of 10 μM NS5806 on I to in SiNC‐ or SiDPP6‐treated hiPSC‐CMs is shown on the right (n = 10, ** P < .01). C 3 , Effect of DPP6 knockdown on the kinetics of I to inactivation in the presence of 10 μM NS5806 in hiPSC‐CMs (* P < .05, ** P < .01 vs SiNC)

Article Snippet: The small interfering RNA (siRNA) duplex against the human DPP6 gene (NM_130797, 5′‐GGTCCATCATCGGCTCTTT‐3′) was designed and constructed by Ribobio (Guangdong, China). hiPSC‐CMs were transfected with siRNA using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instruction at a final siRNA concentration of 100 nM.

Techniques: Expressing, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Inhibition

Journal: The FASEB Journal

Article Title: Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex

doi: 10.1096/fj.201902010RR

Figure Lengend Snippet: Analysis on the putative interactions between KChIP2 and DPP6‐L. A 1 , Modeling and docking simulation of putative interactions between DPP6‐Lin and KChIP2 . Homology model of KChIP2. A 2 , The top‐ranked model of the intracellular domain of DPP6‐L (DPP6‐Lin). A 3 , Top‐ranked models of both proteins. A 4 , Best scored model of docking KChIP2 with DPP6‐Lin; putative‐interacting residues are indicated. B 1 , Schematic depiction of DPP6‐L and the location of mutated residues within the putative KChIP2 interaction site. B 2 , Representative recordings of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut currents from HEK293 cells using 500 ms square voltage pulses (from −40 to +40 mV; holding potential is −80 mV). B 3 , The time constants of inactivation ( τ ) of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut current traces plotted against voltage (** P < .01). C 1 , Representative recordings of I Kv4.3/KChIP2/DPP6‐L‐WT and I Kv4.3/KChIP2/DPP6‐L‐Mut in HEK293 cells before and after 10 μM NS5806. C 2 , Summary data for the effect of NS5806 on the current amplitudes of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut channels (* P < .05). C 3 , Summary data for the effect of NS5806 on the current inactivation kinetics (at +40 mV) of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut channels (** P < .01)

Article Snippet: The small interfering RNA (siRNA) duplex against the human DPP6 gene (NM_130797, 5′‐GGTCCATCATCGGCTCTTT‐3′) was designed and constructed by Ribobio (Guangdong, China). hiPSC‐CMs were transfected with siRNA using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instruction at a final siRNA concentration of 100 nM.

Techniques:

Journal: British Journal of Cancer

Article Title: P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

doi: 10.1038/bjc.2013.484

Figure Lengend Snippet: P2Y2 receptor was significantly expressed in prostate cancer cells. ( A ) mRNA levels of P2Y receptor subtypes were detected by real-time PCR in 2B4, 1E8 and DU-145 cells. The expressions of all P2Y receptor subtypes were normalised by β -actin, and mRNA expression of P2Y1 receptor was defined as 1. ( B ) Protein level of P2Y2 receptor was measured using western blot analysis in prostate cancer cell lines. Results were demonstrated by histograms to quantify the expression levels. Data were presented as mean±s.d. (vertical bars). Three independent experiments were performed.

Article Snippet: P2Y2 shRNA was designed and purchased from Genepharma (Shanghai, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: British Journal of Cancer

Article Title: P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

doi: 10.1038/bjc.2013.484

Figure Lengend Snippet: Effects of P2Y2 receptor knockdown on ATP-mediated in vitro invasion and migration. ( A ) 2B4 and 1E8 cells were transfected with two different P2Y2 siRNAs (siRNA1 and siRNA2) or a control siRNA (NC). Western blotting was used to evaluate the knockdown efficiency. ( B ) Effect of P2Y2 receptor knockdown on in vitro invasion after incubation with ATP for 12 h. ( C ) Effect of P2Y2 receptor knockdown on in vitro migration after incubation with ATP for 12 h. Results were demonstrated by histograms and data were presented as mean±s.d. (vertical bars). Three independent experiments were performed. * P <0.05.

Article Snippet: P2Y2 shRNA was designed and purchased from Genepharma (Shanghai, China).

Techniques: Knockdown, In Vitro, Migration, Transfection, Control, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

doi: 10.1038/bjc.2013.484

Figure Lengend Snippet: Silencing of P2Y2 inhibited the invasion and metastasis of prostate cancer cells in vivo . ( A ) 1E8 cells were stably transfected with P2Y2 shRNA or a scramble shRNA (NC). Two stable P2Y2 shRNA clones (shRNA1 and shRNA2) were shown to express low levels of P2Y2 using western blot analysis. * P <0.05. ( B ) Representative photograph of the tumours in BABL/c nude mice injected with control cells or P2Y2-silenced cells. ( C ) Tumour volume was measured and shown by a histogram. * P <0.05. ( D ) Representative photograph of tumour sections and adjacent tissues (stained with haematoxylin and eosin (HE)). *, tumour mass; N, necrosis; Scale bars=100 μ m. ( E ) Representative photographs of liver section (stained with HE) from tumour-bearing mice. Scale bars=100 μ m. ( F ) The production of liver metastasis and number of micrometastasis per section in tumour-bearing mice. * P <0.05.

Article Snippet: P2Y2 shRNA was designed and purchased from Genepharma (Shanghai, China).

Techniques: In Vivo, Stable Transfection, Transfection, shRNA, Clone Assay, Western Blot, Injection, Control, Staining

Journal: British Journal of Cancer

Article Title: P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

doi: 10.1038/bjc.2013.484

Figure Lengend Snippet: P2Y2 receptor was involved in the ATP-mediated expression of IL-8, Snail and E-cadherin in prostate cancer cells. ( A ) P2Y2-silenced cells and control cells were treated with 100 μ M ATP for 12 h, and cell supernatant was collected for IL-8 protein examination by ELISA assay. Cells were treated with 100 μ M ATP for 12 h, and western blotting was performed to detect the expression of ( B ) Snail and ( C ) E-cadherin. Results were demonstrated by histograms to quantify the expression levels. Data were presented as mean±s.d. (vertical bars). Three independent experiments were performed. * P <0.05.

Article Snippet: P2Y2 shRNA was designed and purchased from Genepharma (Shanghai, China).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: British Journal of Cancer

Article Title: P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

doi: 10.1038/bjc.2013.484

Figure Lengend Snippet: Silencing of P2Y2 receptor affected the expression of Snail, E-cadherin, Claudin-1 and IL-8 in tumour tissues of mice. ( A ) The protein levels of Snail, E-cadherin and Claudin-1 in tumour tissues was examined by immunofluorescence assay. After staining with Snail, E-cadherin or Claudin-1 antibodies, cells were further stained with DAPI. Immunofluorescent images were taken with the confocal microscope. ( B ) The protein levels of Snail, E-cadherin and Claudin-1 in tumour tissues were detected using western blotting. ( C ) IL-8 expression in tumour tissues was examined by ELISA assay. Three separate tumours from three mice were used in the experiments. * P <0.05 vs NC tumours.

Article Snippet: P2Y2 shRNA was designed and purchased from Genepharma (Shanghai, China).

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay