Journal: Frontiers in Pharmacology

Article Title: Salidroside derivative SHPL-49 enhances synaptic remodeling in BCCAO rats via the CDK5/p35/p25 signaling pathway

doi: 10.3389/fphar.2026.1727177

Figure Lengend Snippet: SHPL-49 promotes the expression of CDK5 at both mRNA and protein levels. (A,B) Correlation analysis among samples. (C,D) Differential expression analysis between experimental groups. (E) Venn diagram showing the overlap of differentially expressed genes. (F) GO enrichment analysis of differentially expressed genes. (G–J) qRT-PCR validation of selected differentially expressed genes in primary neurons. (K–N) qRT-PCR validation of differentially expressed genes in rat brain tissue. (O) Representative Western blot images of CDK5 expression in primary neurons. (P) Quantitative analysis of CDK5 protein levels in primary neurons. (Q) Representative Western blot images of CDK5 expression in rat brain tissue. (R) Quantitative analysis of CDK5 protein levels in rat brain tissue. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, OGD/R group vs. Control group, Model group vs. Sham group compared with the Control group, # P < 0.05, ## P < 0.01, ### P < 0.001, SHPL-49 group vs. OGD/R group. n = 3 for mRNA analysis in primary neurons and rat brain tissue. n = 6 for protein analysis in primary neurons and rat brain tissue.

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime, China) in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C with the following primary antibodies: SYP (1:1,000, CST, United States), SYN1 (1:1,000, Boster, China), PSD95 (1:1,000, ABCAM, United States), CDK5 (1:1,000, Boster, China), p35/25 (1:1,000, CST, United States), p-PSD95 (1:1,000, CST, United States).

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Salidroside derivative SHPL-49 enhances synaptic remodeling in BCCAO rats via the CDK5/p35/p25 signaling pathway

doi: 10.3389/fphar.2026.1727177

Figure Lengend Snippet: SHPL-49 inhibits the proteolytic conversion of p35 to p25. (A) Representative Western blot images showing p35 and p25 expression in primary neurons. (B) Quantitative analysis of p35 protein lev-els in primary neurons. (C) Quantitative analysis of p25 protein levels in primary neurons. (D) Representative Western blot images of p35 and p25 in rat brain tissue. (E) Quantitative analysis of p35 protein levels in rat brain tissue. (F) Quantitative analysis of p25 protein levels in rat brain tis-sue. (G) Representative Western blot images showing the expression levels of CDK5, p35, p25, and p-PSD95/PSD95 ratio in primary neurons following OGD/R treatment and co-treatment with SHPL-49 and CDK5 inhibitor Roscovitine. (H) Quantitative analysis of CDK5 protein levels in primary neurons under the same experimental conditions. (I) Quantitative analysis of p35 protein levels in primary neurons under the same experimental conditions. (J) Quantitative analysis of p25 protein levels in primary neurons under the same experimental conditions. (K) Quantitative analysis of p-PSD95/PSD95 ratio in primary neurons under the same experimental conditions. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, OGD/R group vs. Control group, Model group vs. Sham group, # P < 0.05, ## P < 0.01, ### P < 0.001, SHPL-49 group or PD151746 group vs. OGD/R group, SHPL-49 group or SAL group vs. Model group n = 6 per group.

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime, China) in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C with the following primary antibodies: SYP (1:1,000, CST, United States), SYN1 (1:1,000, Boster, China), PSD95 (1:1,000, ABCAM, United States), CDK5 (1:1,000, Boster, China), p35/25 (1:1,000, CST, United States), p-PSD95 (1:1,000, CST, United States).

Techniques: Western Blot, Expressing, Control

Journal: Brain research bulletin

Article Title: Keap1-independent GSK-3β/Nrf2 signaling mediates electroacupuncture inhibition of oxidative stress to induce cerebral ischemia-reperfusion tolerance.

doi: 10.1016/j.brainresbull.2024.111071

Figure Lengend Snippet: Fig. 2. EA pretreatment inhibits oxidative stress and activates the Nrf2 signaling pathway in CIR. (A) Representative images of DHE staining of mouse cerebral cortex, showing red fluorescence for ROS and blue fluorescence for nuclei (n = 3, scale bar: 100 μm). (B) Quantification of ROS fluorescence intensity. Comparison of (C) MDA content, (D) SOD, and (E) Ca2+Mg2+-ATPase activity in brain tissues of mice in each group (n = 3). (F and G) Western blot analysis of protein levels of keap1, Nrf2, HO-1, and NQO1 in ischemic hippocampal tissue (n = 3). Compared with the WT group, **P<0.01, ***P<0.001; compared with the M group, #P<0.05, ##P<0.01, ###P<0.001.

Article Snippet: The membrane was blocked with 5 % skim milk at room temperature for 2 h and then incubated overnight at 4 ◦C with primary antibodies against GSK-3β (BM3904, Boster, Shanghai, China), Keap1 (A00514–3, Boster), Nrf2 (ab137550, Abcam, Cambridge, UK), HO-1 (PB0050, Boster), NQO1 (BM4978, Boster), and GAPDH (BA2913) at a dilution of 1:1000.

Techniques: Staining, Fluorescence, Comparison, Activity Assay, Western Blot

Journal: Brain research bulletin

Article Title: Keap1-independent GSK-3β/Nrf2 signaling mediates electroacupuncture inhibition of oxidative stress to induce cerebral ischemia-reperfusion tolerance.

doi: 10.1016/j.brainresbull.2024.111071

Figure Lengend Snippet: Fig. 4. The essential role of Nrf2 activation in EA protection against CIR in mice. (A) Representative images of DHE staining of mouse cerebral cortex, showing red fluorescence for ROS and blue fluorescence for nuclei (n = 3, scale bar: 100 μm). (B) Quantification of ROS fluorescence intensity. Comparison of (C) MDA content, (D) SOD, and (E) Ca2+Mg2+-ATPase activity in brain tissues of mice in each group (n = 3). (F and G) Western blot analysis of protein levels of keap1, Nrf2, HO-1, and NQO1 in ischemic hippocampal tissue (n = 3). Compared with the EMNrf2(-/-) group, **P<0.01, ***P<0.001; compared with the M group, #P<0.05, ##P<0.01, ###P<0.001.

Article Snippet: The membrane was blocked with 5 % skim milk at room temperature for 2 h and then incubated overnight at 4 ◦C with primary antibodies against GSK-3β (BM3904, Boster, Shanghai, China), Keap1 (A00514–3, Boster), Nrf2 (ab137550, Abcam, Cambridge, UK), HO-1 (PB0050, Boster), NQO1 (BM4978, Boster), and GAPDH (BA2913) at a dilution of 1:1000.

Techniques: Activation Assay, Staining, Fluorescence, Comparison, Activity Assay, Western Blot

Journal: Brain research bulletin

Article Title: Keap1-independent GSK-3β/Nrf2 signaling mediates electroacupuncture inhibition of oxidative stress to induce cerebral ischemia-reperfusion tolerance.

doi: 10.1016/j.brainresbull.2024.111071

Figure Lengend Snippet: Fig. 5. Regulation of the Nrf2 signaling pathway by GSK-3β. HT22 cells were respectively engineered to overexpress, knockdown, and recover GSK-3β. Fluorescence quantitative PCR was used to detect the expression levels of (A) GSK-3β, (B) Nrf2, (C) keap1, (D) HO-1, and (E) NQO1 mRNA (n = 3). (F) Western blot analysis of (G) GSK-3β, (H) Nrf2, (I) keap1, (J) HO-1, and (K) NQO1 protein expression levels (n = 3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The membrane was blocked with 5 % skim milk at room temperature for 2 h and then incubated overnight at 4 ◦C with primary antibodies against GSK-3β (BM3904, Boster, Shanghai, China), Keap1 (A00514–3, Boster), Nrf2 (ab137550, Abcam, Cambridge, UK), HO-1 (PB0050, Boster), NQO1 (BM4978, Boster), and GAPDH (BA2913) at a dilution of 1:1000.

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

Journal: Brain research bulletin

Article Title: Keap1-independent GSK-3β/Nrf2 signaling mediates electroacupuncture inhibition of oxidative stress to induce cerebral ischemia-reperfusion tolerance.

doi: 10.1016/j.brainresbull.2024.111071

Figure Lengend Snippet: Fig. 6. Upregulation of GSK-3β expression attenuates the protective effects of EA against OGD/R-induced neuronal damage. (A) Western blot analysis of (B) GSK-3β, (C) Nrf2, (D) keap1, (E) HO-1, and (F) NQO1 protein expression levels in HT22 cells (n = 3). (G) HT22 cells viability as determined using the CCK-8 assay (n = 6). (H and I) HT22 cell apoptosis as determined by flow cytometry (n = 3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The membrane was blocked with 5 % skim milk at room temperature for 2 h and then incubated overnight at 4 ◦C with primary antibodies against GSK-3β (BM3904, Boster, Shanghai, China), Keap1 (A00514–3, Boster), Nrf2 (ab137550, Abcam, Cambridge, UK), HO-1 (PB0050, Boster), NQO1 (BM4978, Boster), and GAPDH (BA2913) at a dilution of 1:1000.

Techniques: Expressing, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Brain research bulletin

Article Title: Keap1-independent GSK-3β/Nrf2 signaling mediates electroacupuncture inhibition of oxidative stress to induce cerebral ischemia-reperfusion tolerance.

doi: 10.1016/j.brainresbull.2024.111071

Figure Lengend Snippet: Fig. 7. EA pretreatment exerts antioxidative stress effects by regulating the GSK-3β/Nrf2 signaling pathway. (A) Representative images of DHE staining of mouse cerebral cortex, showing red fluorescence for ROS and blue fluorescence for nuclei (n = 3, scale bar: 100 μm). (B) Quantification of ROS fluorescence intensity. Comparison of (C) MDA content, (D) SOD, and (E) Ca2+Mg2+-ATPase activity in brain tissues of mice in each group (n = 3). (F-K) Western blot analysis of GSK-3β, keap1, Nrf2, HO-1, and NQO1 protein expression levels (n = 3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The membrane was blocked with 5 % skim milk at room temperature for 2 h and then incubated overnight at 4 ◦C with primary antibodies against GSK-3β (BM3904, Boster, Shanghai, China), Keap1 (A00514–3, Boster), Nrf2 (ab137550, Abcam, Cambridge, UK), HO-1 (PB0050, Boster), NQO1 (BM4978, Boster), and GAPDH (BA2913) at a dilution of 1:1000.

Techniques: Staining, Fluorescence, Comparison, Activity Assay, Western Blot, Expressing

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Acetylation of CDK5 at K33 causes a loss of kinase activity due to impaired ATP binding. ( a , b ) HEK293 cells were transfected with either FLAG-tagged wild type mouse CDK5 (WT), an acetyl-null mutant (K33R; KR) or a mimetic mutant (K33Q; KQ) of CDK5 in the presence of ( a ) p35-HA or ( b ) p25-HA. Lysates were immunoprecipitated (IPed) with an anti-FLAG antibody and then subjected to an in vitro phosphorylation assay using histone H1 as a substrate. The resulting phosphorylated H1 (P-H1) was visualized via immunoblot analysis (IB) with an anti-phospho-H1 antibody. Coomassie brilliant blue (CBB) staining for H1 was used as a loading control. Immunoprecipitates or whole cell lysates (WCLs) were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for WCL. ( c ) Bacterially purified, recombinant His-tagged CDK5 WT or K33-acetylated CDK5 (Ac-CDK5; Ac) was subjected to an in vitro phosphorylation assay in the presence of H1, [γ- 32 P]ATP and the indicated doses of recombinant p25. The resulting phosphorylated H1 was visualized by autoradiography. Inputs were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. ( d ) Recombinant His-CDK5 WT or His-Ac-CDK5 was incubated with or without resin conjugated to ATP. After washing, the resulting ATP-bound CDK5 was resolved by SDS-PAGE and visualized by IB with an anti-His antibody. Input signals were measured by IB with the indicated antibodies. ( e ) Recombinant His-CDK5 WT (blue-filled circles) or His-Ac-CDK5 (magenta-filled rectangles) was titrated with increasing concentration of mant-ATP. Nonlinear regression was performed to obtain a best-fit curve for a specific binding [Y = Bmax*X/(Kd + X)] and the summary of binding parameters were shown in Supplementary Table . X-axis represents the varying concentration of mant-ATP as indicated. Y-axis represents the relative fluorescence intensity of specific binding, where Bmax is maximum specific binding and Kd is equilibrium binding constant. Wilcoxon matched-pairs rank test was employed to test the binding difference between CDK5 WT and Ac-CDK5 ( ** P = 0.004; Spearman correlation coefficient, rs = 0.976; n = 3). ( f ) Lysates from HEK293 cells expressing p35-FLAG or p25-FLAG were incubated with recombinant His-CDK5 WT or His-Ac-CDK5 bound to Ni-NTA beads. Reaction mixtures were subjected to pull-down and subsequent IB with the indicated antibodies. An anti-FLAG antibody was employed to visualize the extent of CDK5-bound p35 or p25. WCLs were subjected to IB with the indicated antibodies. ( g ) Recombinant His-CDK5 WT plus increasing amounts of recombinant His-Ac-CDK5 was subjected to an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 levels were visualized by autoradiography.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Activity Assay, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, In Vitro, Phospho-proteomics, Western Blot, Staining, Control, SDS Page, Purification, Recombinant, Autoradiography, Incubation, Concentration Assay, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: GCN5 acetylates CDK5 at K33 in the nucleus. ( a ) Lysates obtained from HEK293 cells expressing FLAG-CDK5 plus one of the indicated KAT vectors were subjected to IP with an anti-FLAG antibody followed by IB with an anti-Ac-CDK5 antibody or anti-FLAG antibody. The intensity of the Ac-CDK5 band was measured using Image-J software and normalized to FLAG-CDK5. The fold change over the control (value = 1) is indicated at the bottom of the blot. WCLs were subjected to IB analysis with the indicated antibodies. Each KAT band is marked by the indicated letters. The asterisk indicates non-specific bands. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. *** p < 0.001; * p < 0.05; n.s, not significant. ( b ) Lysates harvested from HEK293 cells expressing FLAG-CDK5 alone or in combination with p35-MYC and/or FLAG-GCN5 were subjected to IP with an anti-FLAG antibody. The bound CDK5 was incubated in the presence of H1 and [γ- 32 P]ATP and visualized by autoradiography. The relative kinase activity of CDK5/p35 was expressed as the fold change over the control (value = 1). The bar represents the mean ± S.D from 3 independent experiments. * p < 0.05. ( c ) HEK293 cells were immunostained with an anti-Ac-CDK5 antibody. Staining specificity was confirmed by pre-incubating with the blocking peptide (EIVAL(acK)RVRLD) that was used to raise the antibody. The nuclei were counterstained with Hoechst dye. Confocal microscopy images are shown. The scale bar represents 10 μm. ( d ) HEK293 cells transfected with the indicated combinations of constructs were subjected to cellular fractionation. The resulting nuclear fractions were IPed with an anti-FLAG antibody and subsequently subjected to either IB with an anti-Ac-CDK5 or anti-FLAG antibody or an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Signals from the phosphorylated H1 were visualized by autoradiography. The fold change over the control (value = 1) is indicated. Nuclear fractions were subjected to IB with the indicated antibodies. Anti-SOD-1 and anti-lamin A/C antibodies were employed to verify the purity of the nuclear fractions.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Expressing, Software, Control, Incubation, Autoradiography, Activity Assay, Staining, Blocking Assay, Confocal Microscopy, Transfection, Construct, Cell Fractionation, In Vitro, Phospho-proteomics

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: SIRT1 is responsible for the deacetylation of Ac-CDK5. ( a ) HEK293 cells transiently expressing FLAG-CDK5 were treated for 24 hrs with increasing doses of nicotinamide (NA, a pan-SIRT inhibitor). Lysates were subjected to IP with an anti-FLAG antibody and probed with the indicated antibodies. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus control (value = 1) was determined. ( b ) HEK293 cells were transfected with one of the FLAG-tagged SIRTs plus FLAG-CDK5 and GCN5-HA. Lysates were subjected to IP with an anti-FLAG antibody and IB with an anti-Ac-CDK5 antibody. WCLs were subjected to IB with the indicated antibodies. Each band of SIRTs is marked by the indicated letters. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. *** p < 0.001; n.s, not significant. ( c ) FLAG-SIRT1 was expressed in HEK293 cells and purified by IP with FLAG beads. SIRT1-bound beads were incubated with recombinant His-Ac-CDK5 supplemented with β-nicotinamide adenine dinucleotide (NAD + ) to activate SIRT1. Reaction mixtures were subjected to IB with an anti-Ac-CDK5 antibody. The fold intensity of Ac-CDK5 versus the control (value = 1) was determined after normalization to the His-Ac-CDK5 inputs. The inputs were probed with the indicated antibodies. ( d , e ) HEK293 cells transfected with FLAG-CDK5 were treated with increasing doses of ( d ) EX527 (a selective SIRT1 inhibitor) or ( e ) SRT1720 (a selective SIRT1 activator) for 24 hrs. Lysates were subjected to IP with an anti-FLAG antibody followed by IB with an anti-Ac-CDK5 antibody. After normalization to FLAG-CDK5, the fold change over the control (value = 1) was determined. ( f ) Lysates were prepared from HEK293 cells transfected with FLAG-CDK5 and p35-HA and exposed to 100 μM EX527 for 24 hrs. Immunoprecipitates purified with an anti-FLAG antibody were subjected either to IB with the indicated antibodies or an in vitro phosphorylation assay in the presence of H1 and cold ATP. Phospho-H1 signals were visualized with an anti-phospho-H1 antibody. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. * p < 0.05.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Expressing, Control, Transfection, Purification, Incubation, Recombinant, In Vitro, Phospho-proteomics

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Temporal expression levels of Ac-CDK5 and SIRT1 are inversely correlated in the rat cortex. ( a ) Three tissue extracts obtained from rat cerebral cortices on embryonic day 17 (E17) and postnatal days 0 (P0), P3, and P7 were subjected to IP with an anti-Ac-CDK5 followed by IB with an anti-CDK5 antibody. CDK5, SIRT1, SIRT2 and GCN5 levels in WCLs were detected by IB with the corresponding antibodies. Blots represent one of three independent experiments. After normalization to GAPDH, relative levels were expressed as the fold intensity over E17 levels (value = 1). The bars represent the mean ± S.D of ( b ) Ac-CDK5, ( c ) SIRT1, ( d ) SIRT2 and ( e ) GCN5. *** p < 0.001; ** p < 0.01; * p < 0.05; n.s, not significant.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Expressing

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Pharmacological modulation of SIRT1 affects Ac-CDK5 levels and kinase activity in hippocampal neurons. ( a ) Primary cultures of hippocampal neurons were prepared from rat hippocampi isolated in gestational day 18. At the indicated days in vitro (DIV), photomicrographs were captured with an Axiovert 100. The scale bar represents 50 μm. ( b ) Cultured hippocampal neurons at DIV5 were fixed and immunostained with an anti-Ac-CDK5 antibody and an anti-NeuN antibody (a neuronal nuclear marker) followed by incubation with appropriate fluorescence-tagged secondary antibodies. Fluorescent images were obtained with an LSM700 confocal microscope. The scale bar represents 10 μm. ( c ) Hippocampal neurons at DIV3 were treated with SRT1720 or EX527 at the indicated doses for 48 hrs. Immunoprecipitates of cellular lysates purified with an anti-CDK5 antibody or IgG were subjected to IB with an anti-CDK5 antibody or an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 signals were visualized by autoradiography. After normalization to H1 or CDK5, the relative intensities of phospho-H1 and Ac-CDK5 were calculated over the controls (value = 1) and are indicated at the bottom of the blot. WCLs were subjected to IB with the indicated antibodies. ( d , e ) The relative kinase activity of CDK5 was expressed as the fold change over the control (value = 1) in the presence of ( d ) SRT1720 or ( e ) EX527. The bar represents the mean ± S.D from 4 independent experiments. *** p < 0.001. ( f ) Hippocampal neurons at DIV3 were treated with EX527 at 100 μM for 48 hrs, fixed, and then processed for immunofluorescent staining as described in ( b ). The scale bar represents 50 μm. ( g ) The fluorescence intensity of Ac-CDK5 in the nuclei of NeuN-positive neurons was measured using Image-J software. The relative fluorescence intensity was expressed as the fold change over the control (value = 1). The bars represent the mean ± S.D of 35 neurons from at least 5 randomly selected areas. *** p < 0.001.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Activity Assay, Isolation, In Vitro, Cell Culture, Marker, Incubation, Fluorescence, Microscopy, Purification, Phospho-proteomics, Autoradiography, Control, Staining, Software

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: CDK5 acetylation hinders neurite outgrowth in hippocampal neurons. ( a ) Hippocampal neurons at DIV3 were transfected with a DsRed-expressing vector containing CDK5 WT or its K33Q mutant (CDK5 KQ) for 48 hrs and subjected to immunofluorescent staining for β-III-tubulin. Fluorescent images were obtained with an LSM700 confocal microscope. The scale bar represents 50 μm. Confocal fluorescent images of ( b ) total neurite length, ( c ) the longest neurite length, ( d ) soma size and ( e ) the number of primary neurites were analyzed using Image-J software. The bars represent the mean ± S.D. of 35–40 neurons from at least 5 randomly selected areas. *** p < 0.001; ns, not significant.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Transfection, Expressing, Plasmid Preparation, Mutagenesis, Staining, Microscopy, Software

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: SIRT1-mediated deacetylation of nuclear CDK5 stimulates neurite outgrowth via upregulation of BDNF transcription. ( a ) Hippocampal neurons at DIV3 were transfected with nuclear localization signal (NLS)-GFP-CDK5 WT or NLS-GFP-K33Q and the DsRed vector. The cultures were then treated with vehicle or 100 nM SRT1720 for 48 hrs, fixed and imaged with a confocal microscope. Other photomicrographs show enlarged views with scale bars of 10 μm. Confocal images of ( b ) total neurite length, ( c ) the length of the longest neurite, ( d ) soma size and ( e ) the number of primary neurites were analyzed using Image-J software. The bars represent the mean ± S.D. based on the measurement of 35–40 neurons from at least 5 randomly selected areas. *** p < 0.001; ** p < 0.01; * p < 0.05; n.s, not significant. The scale bar represents 50 μm. ( f ) Schematic diagram of the rat bdnf gene structure. Bdnf transcription is initiated by multiple promoters located upstream of distinct 5′ exons (white boxes, exon I to VIII) to give rise to transcript variants linked to the 3′ exon (gray box, exon IX). The real-time PCR primers for all bdnf transcripts and transcript containing exon IV are indicated by red and green arrows repectively. The location of the ChIP probe is indicated by a blue bar. ( g , h ) Hippocampal neurons at DIV3 were treated with 100 nM SRT1720 for 24 hrs followed by the addition of 10 μM roscovitine (Ros) for an additional 24 hrs. Total RNA was extracted from the cultures, reverse transcribed and subjected to quantitative real-time PCR for ( g ) all bdnf transcripts and ( h ) transcripts containing exon IV. The values are expressed as the fold-change over the control (value = 1). The bars represent the mean ± S.D. of three independent experiments. ** p < 0.01. ( i ) Total RNA from hippocampal neurons transfected with control siRNA or siRNA against bdnf were analyzed for levels of bdnf transcript by using quantitative real-time PCR. ( j ) The bdnf -knockdowned neurons or control siRNA-transfected neurons were treated with or without 100 nM SRT1720 for 48 hrs. Immunostaining with β-III-tubulin was performed to visualize neurite extension. The scale bar represents 50 μm. ( k ) Confocal fluorescent images of total neurite length were analyzed using Image-J software. The bars represent the mean ± S.D. based on the measurement of 35–40 neurons from at least 5 randomly selected areas. *** p < 0.001; n.s, not significant.

Article Snippet: For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo (Genscript, #SD1211).

Techniques: Transfection, Plasmid Preparation, Microscopy, Software, Real-time Polymerase Chain Reaction, Reverse Transcription, Control, Immunostaining