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: Oxidative Medicine and Cellular Longevity

Article Title: The Isoquinoline Alkaloid Dauricine Targets Multiple Molecular Pathways to Ameliorate Alzheimer-Like Pathological Changes In Vitro

doi: 10.1155/2018/2025914

Figure Lengend Snippet: The primary antibodies used in this study.

Article Snippet: CDK5 , ab40773 , AB_726779 , Rabbit , 1 : 3000 , Abcam.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Isoquinoline Alkaloid Dauricine Targets Multiple Molecular Pathways to Ameliorate Alzheimer-Like Pathological Changes In Vitro

doi: 10.1155/2018/2025914

Figure Lengend Snippet: DAU ameliorated tau pathology via PP2A and p35/25 in both N2a/APP cells and HEK293/Tau cells. Phosphorylation of GSK3 and PP2A and levels of p35/25 and CDK5 in N2a/APP cells (a, b) and HEK293/Tau cells (c, d) as determined by Western blot analysis. β -Actin was used as a loading control. N = 3. Data show the mean ± SEM. ∗∗ P < 0.01 compared to N2a/WT cells, # P < 0.05 and ### P < 0.001 compared to untreated N2a/APP cells, and && P < 0.01 and &&& P < 0.001 compared to untreated HEK293/Tau cells.

Article Snippet: CDK5 , ab40773 , AB_726779 , Rabbit , 1 : 3000 , Abcam.

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Isoquinoline Alkaloid Dauricine Targets Multiple Molecular Pathways to Ameliorate Alzheimer-Like Pathological Changes In Vitro

doi: 10.1155/2018/2025914

Figure Lengend Snippet: The mode of action of DAU. DAU treatment suppressed AD-related changes, notably A β accumulation and tau phosphorylation via APP processing and the CDK5, PP2A, and p35/25 pathways, which may be attributed to the modification of proteins related to functions of oxidative stress, ER stress, molecular chaperones, and signaling protein.

Article Snippet: CDK5 , ab40773 , AB_726779 , Rabbit , 1 : 3000 , Abcam.

Techniques: Modification