1791  (Bio-Techne corporation)

Journal: Aging Cell

Article Title: Periodic Therapeutic Phlebotomy Mitigates Systemic Aging Phenotypes by Promoting Bone Marrow Function

doi: 10.1111/acel.70400

Figure Lengend Snippet: PTP intervention mitigates the decline in neurogenesis and excitability of neurons. (A) Representative immunofluorescence staining of DCX in hippocampus coronal DG of rats (scale bars, 100 μm), and quantification of DCX + cells, n = 3/group. (B) Representative immunofluorescence staining of SOX2 in hippocampus coronal DG of rats (scale bars, 100 μm), and quantification of SOX2 + cells, n = 3/group. (C) Representative immunofluorescence staining of Ki67 and SOX2 in hippocampus coronal SGZ of rats (scale bars, 100 μm), and quantification of Ki67 + SOX2 + cells, n = 3/group. (D) Representative immunofluorescence staining of ASCL1 and SOX2 in hippocampus coronal SGZ of rats (scale bars, 100 μm), and quantification of ASCL1 + SOX2 + cells, n = 3/group. (E) Representative immunofluorescence staining of IL‐6 and SOX2 in hippocampus coronal SGZ of rats (scale bars, 100 μm), and quantification of IL‐6 + SOX2 + cells, n = 3/group. (F) Representative immunofluorescence staining of CDKN2A and SOX2 + in hippocampus coronal SGZ of rats (scale bars, 100 μm), and quantification of CDKN2A + SOX2 + cells, n = 3/group. (G) Representative immunofluorescence staining of CDKN2B and SOX2 in hippocampus coronal SGZ of rats (scale bars, 100 μm), and quantification of CDKN2B + SOX2 + cells, n = 3/group. (H) Representative immunofluorescence staining of p‐γH2A.X and SOX2 in hippocampus coronal SGZ of rats (scale bars, 100 μm), and quantification of p‐γH2A.X + SOX2 + cells, n = 3/group. (I) Normalized fEPSP slope before and after the HFS protocol in the Young (blue), D‐gal (green), and PTP/2w (red) groups of mice. (J) The summary data measured 35–45 min after LTP induction (Young: 194.6% ± 46.94%, n = 13 slices from 4 mice; D‐gal: 119.6% ± 23.75%, n = 13 slices from 5 mice; PTP/2w: 175.1% ± 55.23%, n = 13 slices from 5 mice). (K, L) Cumulative distribution plots and summary of mEPSC amplitude and frequency in the Young (blue), D‐gal (green), and PTP/2w (red) groups of mice in hippocampal CA1 pyramidal cells (Frequency: 3.85 ± 1.8 Hz of Young, n = 17 slices from 4 mice; 1.34 ± 1.11 Hz of D‐gal, n = 16 slices from 5 mice; 2.84 ± 2.02 Hz of PTP/2w, n = 19 slices from 5 mice; Amplitude: 10.50 ± 2.49 pA of Young, n = 17 slices from 4 mice; 10.41 ± 3.26 of D‐gal, n = 16 slices from 5 mice; 10.28 ± 2.12 of PTP/2w, n = 19 slices from 5 mice). (M) Upper: Superimposed representative averaged fEPSCs recorded 10 min before (1) and 35–45 min after (2) LTP induction in the Young (blue), D‐gal (green), and PTP/2w (red) groups of mice. Below: Example traces of mEPSCs measured in CA1 hippocampal pyramidal cells from the Young (blue), D‐gal (green), and PTP/2w (red) groups of mice. (N) Number of intersections by radial distance from soma at hippocampus, 15 neurons from 3 samples. (O) Number of intersections by radial distance from soma at cortex, 15 neurons from 3 samples. Statistical analysis: One‐way ANOVA and Bonferroni post‐test for (I–L) and Tukey post‐test for the others. Data are mean ± SD; error bars denote 95% CI; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The slides were then incubated overnight at 4°C with primary antibodies, specifically targeting DCX (RD, ab207175), SOX‐2 (RD, AF2018), Ki67 (Novus, NBP2‐22112SS), ACSL1 (Affinity, DF8504), GFAP (Affinity, DF6040), p‐γH2A.X (Abclonal, AP0687), CDKN2A (Santa Cruz, Sc‐1661), and CDKN2B (Invitrogen, PA5‐49749) and IL‐6 (Abclonal A21264).

Techniques: Immunofluorescence, Staining

Journal: Journal of Advanced Research

Article Title: Dimeric guaianolide sesquiterpenoids from the flowers of Chrysanthemum indicum ameliorate hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis

doi: 10.1016/j.jare.2024.12.047

Figure Lengend Snippet: Chryindicolide O ( 15 ) directly bound SIRT1 protein and inhibited its ubiquitination degradation. The total SIRT1 protein levels in AML12 cells were examined using Western blotting (A). ITC titration of 15 (200 µM) into recombinant SIRT1 protein (10 µM) (B). DARTs was performed in P/O induced AML12 cells treated with 15 at 10 μM (C). CETSA was carried out after AML12 cells (D) and lysates (E) treated with 15 at 10 μM. The SIRT1 levels in AML12 cells treated with CHX for 0, 1, 2, 4, and 8 h with or without pre-incubation with 15 (10 µM) (F). The ubiquitination of SIRT1 in AML12 cells was examined using Western blotting (G). The mRNA expression of SIRT1 in NAFLD patients utilizing the NCBI GEO dataset that is accessible to the public (H). Stereo view of the binding between the N-terminal sirtuin-activating compound binding domain and 15 using the best mode as representation. The surfaces of the protein and the ligand (displayed in dots) are colored by elements. The figure was prepared in PyMOL (I). Interaction diagram between 15 and SIRT1 (PDB ID: 4ZZJ) analyzed in Maestro 13.6. (J). Contacts between 15 and I223 as well as I227 on SIRT1 (PDB ID: 4ZZJ) analyzed in Maestro 13.6 (K). Superimposition of the docked 4TQ conformation to the original crystal structure (RMSD: 2.65 Å) (L). Interaction diagram between 4TQ and SIRT1 (PDB ID: 4ZZJ) analyzed in Maestro 13.6. (M). β-actin was employed as a loading reference in Western blotting analysis.

Article Snippet: Mammalian expression of flag-tagged SIRT1 plasmid (#1791) was obtained from Addgene.

Techniques: Ubiquitin Proteomics, Western Blot, Titration, Recombinant, Incubation, Expressing, Binding Assay

Journal: Journal of Advanced Research

Article Title: Dimeric guaianolide sesquiterpenoids from the flowers of Chrysanthemum indicum ameliorate hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis

doi: 10.1016/j.jare.2024.12.047

Figure Lengend Snippet: Chryindicolide O ( 15 ) inhibited SIRT1-mediated lipid aggravation in P/O induced AML12 cells. Nile red (A) and Bodipy 493/503 (B) staining in P/O induced AML12 cells treated with 15 (10 µM), with or without pre-incubation with EX-527. TG (C), T-CHO (D), NEFA (E) content in P/O induced AML12 cells treated with 15 (10 µM), with or without pre-incubation with EX-527. The acetylated, total, and nuclear FoxO1 protein content in AML12 cells were examined using Western blotting (F). Immunofluorescent images of FoxO1 protein in AML12 cells (G). The SREBP1, ACLY, ACC, FASN, and SCD1 protein levels in lysates and SREBP1 in nucleus of AML12 cells were analyzed by Western blotting (H). Immunofluorescent images of SREBP1 protein in AML12 cells (I). The acetylated and total PGC-1α protein changes in AML12 cells were examined using Western blotting (J). The levels of co-precipitated PGC-1α with PPARα, total and nuclear PPARα in AML12 cells were examined using Western blotting (K). The CPT1A protein level in AML12 cells were examined using Western blotting (L). β-actin was employed as a loading reference in lysates, and histone H3 served as a loading standard in nucleus in Western blotting analysis. Data are presented as means ± SD. n = 3. ## P < 0.01, P/O vs. vehicle; ** P < 0.01, P/O + 15 vs. P/O; & P < 0.05 and && P < 0.01, P/O + EX-527 vs. P/O; $ P < 0.05, P/O + EX-527 + 15 vs. P/O + EX-527. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mammalian expression of flag-tagged SIRT1 plasmid (#1791) was obtained from Addgene.

Techniques: Staining, Incubation, Western Blot

Journal: Journal of Advanced Research

Article Title: Dimeric guaianolide sesquiterpenoids from the flowers of Chrysanthemum indicum ameliorate hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis

doi: 10.1016/j.jare.2024.12.047

Figure Lengend Snippet: Chryindicolide O ( 15 ) inhibited SIRT1-mediated ferroptosis in P/O induced AML12 cells. The GPX4 protein level in P/O induced AML12 cells subjected to varying doses of 15 (A). The GPX4 protein level in P/O induced AML12 cells subjected to 15 (10 µM), in the presence or absence of pre-incubation with EX-527 (B). Relative intracellular Fe 2＋ level were measured by ferroOrange in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (C). Relative lipid peroxidation level was measured by liperfluo in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (D). Relative intracellular ROS level was measured by DCFH-DA in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (E). GSH level (F), SOD activity (G), and MDA content (H) in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527. Relative mitochondrial ROS level was measured by mitoSOX red in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (I). Relative mitochondria content was measured by mitotracker in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (J). Relative mitochondrial function was measured by JC-1 in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (K). ATP content in P/O induced AML12 cells subjected to varying doses of 15 , in the presence or absence of pre-incubation with EX-527 (L). β-actin was employed as a loading reference in Western blotting analysis. Data are presented as means ± SD. n = 3. ## P < 0.01, P/O vs. vehicle; * P < 0.05 and ** P < 0.01, P/O + 15 vs. P/O; & P < 0.05 and && P < 0.01, P/O + EX-527 vs. P/O. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mammalian expression of flag-tagged SIRT1 plasmid (#1791) was obtained from Addgene.

Techniques: Incubation, Activity Assay, Western Blot