Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Effect of Sirtuin-1 on Synaptic Plasticity in Nucleus Accumbens in a Rat Model of Heroin Addiction

doi: 10.12659/MSM.910550

Figure Lengend Snippet: Cells positive for SIRT1, Cdk5, NF-κB, FOXO1, Cbp, and Syn in the NAc region (×400) and their optical density values ( A ) Saline control (SC) group; ( B ) Heroin-addicted (HA) group; ( C ) AAV-rSirt1 group; ( D ) AAV-rSirt1 shRNA group. # P <0.05 compared with the SC group; * P <0.05 compared with the HA group.

Article Snippet: The reagents and analysis systems used were as follows: heroin (purity 92.09%, provided by the Guizhou Public Security Bureau), pentobarbital sodium (Sigma, USA), naloxone hydrochloride (Chongqing YaoPharma, China), mouse monoclonal anti-SIRT1 (Abcam, USA), rabbit polyclonal anti-Cdk5, rabbit polyclonal anti-NF-κB (Bioss, China), rabbit monoclonal anti-FOXO1 (Abcam, USA), rabbit polyclonal anti-Cbp (Bioss, China), rabbit monoclonal anti-PSD95 (Abcam, USA), rabbit monoclonal anti-Syn (Abcam, USA), mouse monoclonal anti-beta-actin (Abcam, USA), SuperScript III RT reverse transcription kit (Invitrogen, USA) Sybr qPCR mix (Invitrogen, USA), Plasmid Mini kit (TransGen Biotech, Beijing, China), gel extraction kit (Omega), Trans2K Plus II DNA Marker (TransGen, Beijing, China), DNA primers (Invitrogen, USA), DNA sequencing (Invitrogen, USA), restriction endonuclease EcoRI (NEB, USA), T4 DNA Ligase (NEB, USA), Plasmid Maxi Kit (Qiagen, Germany), CPT High-efficiency Transfection Kit (Virotherapy Technologies, Wuhan, China).

Techniques: shRNA

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Effect of Sirtuin-1 on Synaptic Plasticity in Nucleus Accumbens in a Rat Model of Heroin Addiction

doi: 10.12659/MSM.910550

Figure Lengend Snippet: Detection of SIRT1, Cdk5, FOXO1, NF-κB, Syn, and PSD95 protein levels in each group by immunoblotting. # P <0.05 compared with the saline control group; * P <0.05 compared with the HA group .

Article Snippet: The reagents and analysis systems used were as follows: heroin (purity 92.09%, provided by the Guizhou Public Security Bureau), pentobarbital sodium (Sigma, USA), naloxone hydrochloride (Chongqing YaoPharma, China), mouse monoclonal anti-SIRT1 (Abcam, USA), rabbit polyclonal anti-Cdk5, rabbit polyclonal anti-NF-κB (Bioss, China), rabbit monoclonal anti-FOXO1 (Abcam, USA), rabbit polyclonal anti-Cbp (Bioss, China), rabbit monoclonal anti-PSD95 (Abcam, USA), rabbit monoclonal anti-Syn (Abcam, USA), mouse monoclonal anti-beta-actin (Abcam, USA), SuperScript III RT reverse transcription kit (Invitrogen, USA) Sybr qPCR mix (Invitrogen, USA), Plasmid Mini kit (TransGen Biotech, Beijing, China), gel extraction kit (Omega), Trans2K Plus II DNA Marker (TransGen, Beijing, China), DNA primers (Invitrogen, USA), DNA sequencing (Invitrogen, USA), restriction endonuclease EcoRI (NEB, USA), T4 DNA Ligase (NEB, USA), Plasmid Maxi Kit (Qiagen, Germany), CPT High-efficiency Transfection Kit (Virotherapy Technologies, Wuhan, China).

Techniques: Western Blot

Journal: bioRxiv

Article Title: ACSS2 regulates ferroptosis in an E2F1-dependent manner in breast cancer brain metastatic cells

doi: 10.1101/2024.10.18.619082

Figure Lengend Snippet: (A) Cell lysate from parental and brain trophic MDA-MB-231, 4T1 and E0771 cells were collected for immunoblot analysis with the indicated antibodies. (B) Cell lysates from MDA-MB-231Br cells expressing shRNA against Scramble or OGT were collected for immunoblot analysis with the indicated antibodies. (C) Representative images of bioluminescent (top) detection of tumors from mice injected with shSCR and shOGT MDA-MB-231BR cells 14 days post-injection. Representative images of H&E analysis (bottom) on coronal sections from mice harboring shRNA against Scramble or OGT tumors at Day 14. Data are quantified and presented as average Bioluminescence signal from mice injected with MDA-MB-231BR cells expressing shRNA against Scramble (n=3) or OGT mice (n=3) (bottom). Student’s t-test reported as mean ± SEM; **p<0.001. (D) Cell lysates from MDA-MB-231Br cells with shRNA against Scramble or CDK5 were collected for immunoblot analysis with the indicated antibodies. (E) Representative images of bioluminescent (top) detection of tumors from mice injected with shScramble and shCDK5 MDA-MB-231BR cells 14 days post-injection. Representative images of H&E analysis (bottom) on coronal sections from mice harboring shRNA against Scramble or CDK5 tumors at Day 14. Data are quantified and presented as average Bioluminescence signal from mice injected with MDA-MB-231BR cells expressing shSCR (n=3) or shCDK5 mice (n=3) (bottom). Student’s t-test reported as mean ± SEM; *p<0.05.

Article Snippet: Anti-actin (Santa Cruz Biotechnology; Dallas, TX, USA), anti-OGT, anti-ACSS2, anti-Cdk5, (Cell Signaling Technology; Danvers, MA, USA), anti-O-GlcNAc (Sigma Aldrich; St. Louis, MO, USA) Anti-E2F1, Anti-GPX4, Anti-SLC7A11 (Abcam).

Techniques: Western Blot, Expressing, shRNA, Injection

Journal: bioRxiv

Article Title: ACSS2 regulates ferroptosis in an E2F1-dependent manner in breast cancer brain metastatic cells

doi: 10.1101/2024.10.18.619082

Figure Lengend Snippet: (A) Cell lysates from MDA-MB-231BR cells stably expressing shRNA against Scramble or E2F1 were collected for immunoblot analysis with the indicated antibodies. (B) Total RNA was collected from MDA-MB-231BR cells stably expressing shRNA against Scramble or E2F1. Quantification of qRT-PCR performed on RNA extracts analyzing E2F1, SLC7A11, and GPX4 gene expression normalized to GAPDH. Two-way ANOVA reported as mean ± SEM.* p-value < 0.05. (C) Cell lysates from 4T1BR cells stably expressing shRNA against Scramble or E2F1 were collected for immunoblot analysis with the indicated antibodies. (D) Cell lysates from MDA-MB-231BR cells stably expressing shRNA against Scramble or ACSS2 were collected for immunoblot analysis with the indicated antibodies . (E) Cell lysates from 4T1-BR cells stably expressing shRNA against Scramble or ACSS2 were collected for immunoblot analysis with the indicated antibodies. (F) Cell lysates from E0771-BR cells stably expressing shRNA against Scramble or ACSS2 were collected for immunoblot analysis with the indicated antibodies. (G) Cell lysates from MDA-MB-231 cells stably overexpressing Control or OGT were collected for immunoblot analysis with the indicated antibodies . (H) Cell lysates from MDA-MB-231 cells stably overexpressing Control or CDK5 were collected for immunoblot analysis with the indicated antibodies. (I) Cell lysates from MDA-MB-231 cells stably overexpressing Control or HA-ACSS2-S267D were collected for immunoblot analysis with the indicated antibodies.

Article Snippet: Anti-actin (Santa Cruz Biotechnology; Dallas, TX, USA), anti-OGT, anti-ACSS2, anti-Cdk5, (Cell Signaling Technology; Danvers, MA, USA), anti-O-GlcNAc (Sigma Aldrich; St. Louis, MO, USA) Anti-E2F1, Anti-GPX4, Anti-SLC7A11 (Abcam).

Techniques: Stable Transfection, Expressing, shRNA, Western Blot, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: ACSS2 regulates ferroptosis in an E2F1-dependent manner in breast cancer brain metastatic cells

doi: 10.1101/2024.10.18.619082

Figure Lengend Snippet: (A) Cell lysates from MDA-MB-231BR cells treated with control (DMSO) or ACSS2 inhibitor AD-5584 (100 μM) for 48 hrs were collected for immunoblot analysis with the indicated antibodies. ( B) Quantification of propidium iodine staining flow cytometry of MDA-MB-231BR cells treated with DMSO, AD-5584, or AD-5584 + Fer1. Data reported as One-way ANOVA with Tukey’s multiple comparisons test reported as mean ± SEM *p-value < 0.05, **p<0.01. (C) Quantification of lipid peroxides with Bopidy C11 and flow cytometry of MDA-MB-231BR (right) cells treated with DMSO, AD-5584, or AD-5584 + Fer1. Data reported as One-way ANOVA with Tukey’s multiple comparisons test reported as mean ± SEM **p-value < 0.01. (D) Quantification of propidium iodine staining flow cytometry of MDA-MB-231BR cells overexpressing control or E2F1 treated with DMSO, AD-5584, or AD-5584. Data reported as One-way ANOVA with Tukey’s multiple comparisons test reported as mean ± SEM *p-value < 0.05, **p<0.01 (E) Quantification of lipid peroxides with Bopidy C11 and flow cytometry of MDA-MB-231BR cells overexpressing E2F1 treated with DMSO, AD-5584, or AD-5584. Data reported as One-way ANOVA with Tukey’s multiple comparisons test reported as mean ± SEM **p-value < 0.01, ***p-value < 0.001. (F) Representative images depicting tumor growth in organotypic brain slices derived from mice intracranially injected with MDA-MB-231Br-luc cells detected via bioluminescence. Brain slices containing tumors are treated with Control (DMSO), AD-5584 or AD-5584 and Ferrostatin-1 (Fer-1) for indicated days (left). Quantification of tumor Bioluminescence at indicated day (right) (Control: DMSO n=3, AD-5584 n=3, AD-5584 +Fer-1 n=3) One-way ANOVA with Tukey’s multiple comparisons test reported as mean ± SEM *p-value < 0.05, **p<0.01. (G) Representative images of bioluminescent detection of tumors from Balb/C mice injected with luciferase-tagged 4T1BR cells at Day 0 (prior to drug treatment) and at 10 days post-drug treatment. Data are quantified and presented as average Relative Bioluminescence signal from mice injected with 4T1BR cells treated with Vehicle (n=3) or AD-5584 treated mice (n=3) (right). Student’s t-test reported as mean ± SEM; *p<0.05. Representative images of brain sections stained for H&E 10-days-post treatment. (H) Kaplan-Meyer survival analysis quantifying survival of mice injected with 4T1BR cells and treated with vehicle (n=5) or AD-5584 (n=5), *p<0.05. (I) Working model schematic depicting OGT regulates ACSS2 via CDK5 phosphorylation at Serine-267 residue. BCBM cells upregulate ACSS2 to convert acetate to acetyl-CoA. Acetyl-CoA promotes the transcription of E2F1, leading to the transcription of anti-ferroptotic E2F1 target genes, SLC7A11 and GPX4. Expression of SLC7A11and GPX4 protects BCBM cells from ferroptosis.

Article Snippet: Anti-actin (Santa Cruz Biotechnology; Dallas, TX, USA), anti-OGT, anti-ACSS2, anti-Cdk5, (Cell Signaling Technology; Danvers, MA, USA), anti-O-GlcNAc (Sigma Aldrich; St. Louis, MO, USA) Anti-E2F1, Anti-GPX4, Anti-SLC7A11 (Abcam).

Techniques: Control, Western Blot, Staining, Flow Cytometry, Derivative Assay, Injection, Luciferase, Residue, Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Surgical Stress Induces Brain‐Derived Neurotrophic Factor Reduction and Postoperative Cognitive Dysfunction Via Glucocorticoid Receptor Phosphorylation in Aged Mice

doi: 10.1111/cns.12368

Figure Lengend Snippet: Surgical stress elevated GR phosphorylation at ser220 by CDK5 and its regulator in prefrontal cortex of POCD mice. (A), GR phosphorylation was tested with Western blot. The phosphorylation at ser220 site was significantly elevated in POCD group (two‐tailed unpaired t‐test, P = 0.0136 for pGRs220; P = 0.2303 for pGRs212; P = 0.3860 for pGRs234), compared with control group. (B) The level of CDK5 and its regulators p35, p25 in prefrontal cortex was tested with Western blot in POCD and control mice. The level of CDK5 was upregulated in POCD group compared with control (two‐tailed unpaired t‐test, P = 0.0178). CDK5 regulators p35 (P = 0.0140), p25 (P = 0.0139) also elevated in prefrontal cortex compared with control. (C), nonparametric correlation analysis of GR phosphorylation and behavioral index in 20‐month groups (CDK5 with MWM, r2 = 0.75, P < 0.001; CDK5 with EPM, r2 = 0.78, P < 0.001; pGRs220 with MWM, r2 = 0.63, P < 0.001; pGRs220 with EPM, r2 = 0.73, P < 0.001) (C). (*P < 0.05, POCD compared with control) (n = 8 mice/group). Error bar represents SEM. (D), no significant changes on pGRs220 (P = 0.7576), CDK5 (P = 0.8640) and its regulators (P = 0.8182 for p35 and P = 0.8844 for p25) in 6‐month groups. (*P < 0.05, POCD compared with control) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Article Snippet: After transferred onto PVDF membrane, the following primary antibodies were used: sheep anti‐BDNF antibody (1:5000, millipore, Billerica, MA, USA); rabbit anti‐GR and anti‐p‐GRs220 antibody (1:12000, CST, Boston, MA, USA); rabbit anti‐CDK5 antibody (1:5000, BD, NJ, USA); rabbit anti‐p35 antibody (1:5000, BD); rabbit anti‐p‐GRs212 (1:2000, Anbo, San Francisco, CA, USA); and rabbit anti‐p‐GRs234 (1:12000, CST).

Techniques: Western Blot, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Surgical Stress Induces Brain‐Derived Neurotrophic Factor Reduction and Postoperative Cognitive Dysfunction Via Glucocorticoid Receptor Phosphorylation in Aged Mice

doi: 10.1111/cns.12368

Figure Lengend Snippet: Surgical stress‐induced GR phosphorylation, BDNF reduction and cognitive dysfunction are rescued by CDK5 inhibitor. (A) Roscovitine, a specific CDK5 inhibitor was treated after the surgery. The performance on Morris water maze and elevated plus maze was rescued with treatment of roscovitine. POCD+roscovitine group performed shorter latency to find the hidden platform (two‐way ANOVA, treatment condition × day, F(1,98) = 11.21, P < 0.001; POCD+roscovitine compared with POCD group followed by Tukey's post hoc test, P < 0.001; one‐way ANOVA following with Tukey's post hoc test was used to compare latency on each day, P < 0.05 on day 6 and 7, POCD+roscovitine compared with POCD) and spent longer time in target area with a removed platform (one‐way ANOVA following with Tukey's post hoc test, P = 0.0446), compared with POCD group. No significant difference on swimming speed among groups (P = 0.8075, POCD+roscovitine compared with POCD) and swimming paths were also presented. In elevated plus maze test, time spent in open arm was also rescued with roscovitine treatment (one‐way ANOVA following with Tukey's post hoc test, P = 0.0199, POCD+roscovitine compared with POCD). (B), the level of GR phosphorylation at ser220 site in nucleus was rescued in POCD+roscovitine group compared with POCD (P = 0.0277). The expression of BDNF level in prefrontal cortex was also rescued in POCD+roscovitine group compared with POCD (P = 0.0162). (*P < 0.05, POCD compared with control; # P < 0.05, POCD+roscovitine compared with POCD) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Article Snippet: After transferred onto PVDF membrane, the following primary antibodies were used: sheep anti‐BDNF antibody (1:5000, millipore, Billerica, MA, USA); rabbit anti‐GR and anti‐p‐GRs220 antibody (1:12000, CST, Boston, MA, USA); rabbit anti‐CDK5 antibody (1:5000, BD, NJ, USA); rabbit anti‐p35 antibody (1:5000, BD); rabbit anti‐p‐GRs212 (1:2000, Anbo, San Francisco, CA, USA); and rabbit anti‐p‐GRs234 (1:12000, CST).

Techniques: Expressing