Journal: Cell Death & Disease

Article Title: The NR3C2-SIRT1 signaling axis promotes autophagy and inhibits epithelial mesenchymal transition in colorectal cancer

doi: 10.1038/s41419-025-07575-3

Figure Lengend Snippet: A Volcano plot of downstream regulatory gene after NR3C2 overexpression. B TIMER prediction of the relationship between NR3C2 and SIRT1. left: COAD; right: READ; C Western blot detection of SIRT1 expression after NR3C2 overexpression and NR3C2 knockdown, * P < 0.05, ** P < 0.01; D Fluorescence double staining to detect the expression of NR3C2 and SIRT1 (Blue: DAPI; Red: NR3C2; Green: SIRT1). Upper: HCT116, lower: SW620; E ChIP assay to detect the relationship between NR3C2 and SIRT1. * P < 0.05. F Transwell migration experiment validating that siSIRT1 increases the decrease in cells migration. Upper: HCT116, lower: RKO, right: statistical chart, * P < 0.05(HCT116), # P < 0.05(RKO).

Article Snippet: Cell lysates were incubated with antibodies (anti-SIRT1 antibody or anti-NR3C2 antibody or control IgG), followed by incubation with protein A/G magnetic beads (MCE, USA) at 4 °C overnight.

Techniques: Over Expression, Western Blot, Expressing, Knockdown, Fluorescence, Double Staining, Migration

Journal: Cell Death & Disease

Article Title: The NR3C2-SIRT1 signaling axis promotes autophagy and inhibits epithelial mesenchymal transition in colorectal cancer

doi: 10.1038/s41419-025-07575-3

Figure Lengend Snippet: A Photograph of mouse lung metastasis model using RKO cells, right: weight change of mice; B HE staining of mouse lung tissues for RKO-vector and RKO-NR3C2; C Photograph of mouse lung metastasis model using HCT116 cells, right: weight change of mice; D HE staining of mouse lung tissues for HCT116-vector and HCT116-NR3C2; E Western blot detection of NR3C2, SIRT1, and P62 expression in the lung tissues of mice in the RKO group, * P < 0.05; F Western blot detection of NR3C2, SIRT1, and P62 expression in the lung tissues of mice in the HCT116 group, * P < 0.05; G IHC detection of NR3C2 expression in mouse lung tissues (50X and 400X); H IHC detection of SIRT1 expression in mouse lung tissues(50X and 400X).

Article Snippet: Cell lysates were incubated with antibodies (anti-SIRT1 antibody or anti-NR3C2 antibody or control IgG), followed by incubation with protein A/G magnetic beads (MCE, USA) at 4 °C overnight.

Techniques: Staining, Plasmid Preparation, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: The NR3C2-SIRT1 signaling axis promotes autophagy and inhibits epithelial mesenchymal transition in colorectal cancer

doi: 10.1038/s41419-025-07575-3

Figure Lengend Snippet: NR3C2 and SIRT1 constitute a pivotal signaling axis that modulates EMT in CRC cells through the intricate mechanism of autophagy.

Article Snippet: Cell lysates were incubated with antibodies (anti-SIRT1 antibody or anti-NR3C2 antibody or control IgG), followed by incubation with protein A/G magnetic beads (MCE, USA) at 4 °C overnight.

Techniques:

Journal: Clinical immunology (Orlando, Fla.)

Article Title: Metabolic Reprogramming in Memory CD4 T Cell Responses of Old Adults

doi: 10.1016/j.clim.2019.07.003

Figure Lengend Snippet: (A) AMPK phosphorylation was compared by Peggy Sue western in day 2 activated CD4 memory T cells from 12 young and 12 older adults. Representative blot of signal intensities (left) and summary data (right). Comparison by unpaired one-tailed t-test. (B) Activated CD4 T memory cells were transfected with siRNA for AMPKα on day 1. Knockdown efficiency by qPCR (left) and CPT1a expression by Peggy Sue western (right) are shown on day 2 after activation (n=9). (C) SIRT1 expression in day 2 activated CD4 memory T cells were determined by Peggy Sue western. A representative blot of signal intensities from a young and an old adult (left) and the ratio of SIRT1 to actin from the samples shown in (A) are shown. (D) Activated CD4 T memory cells were transfected with siRNA for SIRT1 on day 1. Knockdown efficiency by Peggy Sue western (left) and CPT1a expression by qPCR (middle) and Peggy Sue western (right) are shown on day 2 after activation (n=6). Comparisons were done by paired one-tailed t-test.

Article Snippet: The high sensitivity protocol was applied by increasing the stacking loading time to 20 seconds, the sample loading time to 12 seconds, and the separation time to 45 min. Primary antibodies were as listed: CPTIa (Cell Signaling, Cat#: 12252), SIRT1 (Novus Biologicals, Cat#: NBP1–49540), AMPK (R&D, Cat#: MAB3197), phospho-AMPK (Cell Signaling, Cat#: 2535), Cytochrome c (Cell Signaling, Cat#: 11940).

Techniques: Phospho-proteomics, Western Blot, Comparison, One-tailed Test, Transfection, Knockdown, Expressing, Activation Assay

Journal: NPJ Aging

Article Title: Pyrroloquinoline quinone (PQQ) protects mitochondrial function of HEI-OC1 cells under premature senescence

doi: 10.1038/s41514-022-00083-0

Figure Lengend Snippet: A SIRT1 and PGC-1α protein expression was analyzed by Western blotting (upper, middle) using β-actin as a loading control (lower). B Relative protein expression level of SIRT1 normalized by β-actin. n = 10 per group. C Relative protein expression level of PGC-1α normalized by β-actin. D Acetylation of PGC-1α analyzed by immunoprecipitation. E Relative acetylation level of PGC-1α normalized by PGC-1α. F Relative mRNA expressions of GAPDH, SIRT1, and PGC-1α using β-actin as an internal control. G The changes of SIRT1 expression in flow cytometry analysis. H The rate of SIRT1 positive cells ( B , C , E , H : n = 5 per group, F : n = 3 per group; repeated experiments). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. RQ data are shown as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ***** P < 0.00001.

Article Snippet: In the staining of SIRT1, the cells were washed with DPBS, harvested from the flasks via trypsinization (0.05% trypsin, 0.53 mM EDTA for 2 min), fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) (10010023; Thermo Fisher Scientific Inc., USA) at room temperature for 10 min, washed with PBS, incubated in 0.1% Triton X-100 in PBS at room temperature for 5 min, washed with PBS two times, stained with Alexa Fluor 488 Phalloidin (12:1000, A12379; Thermo Fisher Scientific Inc., USA) and anti-SIRT1 antibody (1:500, NBP1-51641AF647 (1.77 mg/mL), Novus Biologicals, Inc.) in PBS with 0.5% BSA at room temperature for 30 min, washed with PBS two times and diluted in PBS for flow cytometry analysis.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Flow Cytometry, Standard Deviation

Journal: NPJ Aging

Article Title: Pyrroloquinoline quinone (PQQ) protects mitochondrial function of HEI-OC1 cells under premature senescence

doi: 10.1038/s41514-022-00083-0

Figure Lengend Snippet: A Sirtuin 1 (SIRT1) expression and deacetylation activity are decreased in HEI-OC1 cells by H 2 O 2 exposure. PGC-1α mediates the protective effect of SIRT1 expression and mitochondrial biogenesis and function. PQQ improves mitochondrial biogenesis and function by restoring SIRT1 expression and deacetylation activity under H 2 O 2 exposure in HEI-OC1 cells. B Schematic diagram of mitochondrial morphological changes.

Article Snippet: In the staining of SIRT1, the cells were washed with DPBS, harvested from the flasks via trypsinization (0.05% trypsin, 0.53 mM EDTA for 2 min), fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) (10010023; Thermo Fisher Scientific Inc., USA) at room temperature for 10 min, washed with PBS, incubated in 0.1% Triton X-100 in PBS at room temperature for 5 min, washed with PBS two times, stained with Alexa Fluor 488 Phalloidin (12:1000, A12379; Thermo Fisher Scientific Inc., USA) and anti-SIRT1 antibody (1:500, NBP1-51641AF647 (1.77 mg/mL), Novus Biologicals, Inc.) in PBS with 0.5% BSA at room temperature for 30 min, washed with PBS two times and diluted in PBS for flow cytometry analysis.

Techniques: Expressing, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 3. ZIP8-dependent zinc metabolism regulates AEC2 renewal through SIRT1. (A) IPA pathway analysis of AEC2s from healthy and IPF lungs.(B) Sir- tuin activation score of healthy and IPF AEC2s. (C and D) SIRT1 expression in freshly isolated AEC2s (C) and AEC2s derived from 3D organoids (D) by qPCR (n = 4–6, *P < 0.05). (E) SIRT1 expression of AEC2s derived from 3D-cultured organoids of ZIP8+ and ZIP8– AEC2s (n = 5 each, *P < 0.05). (F) Intracellular SIRT1 levels in healthy and IPF AEC2s without and with ZnSO4 treatment by flow cytometry (n = 3–4). (G) Intracellular SIRT1 levels in gated ZIP8+ and ZIP8– AEC2s from healthy lungs (n = 4). (H and I) CFE with 3D organoid culture of AEC2s from healthy lungs treated with either 1 μM SRT1720 (n = 4–5, ***P < 0.001 by ANOVA) (H) or 125 and 200 μM splitomicin (n = 3, ***P < 0.001, ****P < 0.0001 by ANOVA) (I). (J) CFE of AEC2s from IPF lungs cultured with SRT1720 at the indicated concentrations (n = 3, **P < 0.01, ****P < 0.0001 by ANOVA). (K) CFE of AEC2s from IPF lungs cultured with 1 μM SRT1720, 100 μM ZnSO4, or both (n = 3–4, ****P < 0.0001 by ANOVA). (L–N) A549 cells with SIRT1 knockout and control cells. SIRT1 ko 1, set 1 sgRNA; SIRT ko 2, set 2 sgRNA. (L) SIRT1 expression by qPCR. (M) SIRT1 expression by Western blot analysis; the same experiments were performed 3 times. (N) CFE with 3D organoid culture (n = 4, ***P < 0.001 by ANOVA). Data are shown as mean ± SEM. Unpaired 2-tailed Student’s t test.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Activation Assay, Expressing, Isolation, Derivative Assay, Cell Culture, Flow Cytometry, Knock-Out, Control, Western Blot

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 4. Downregulated ZIP8/SIRT1 signaling and decreased renewal capacity of AEC2s from old mouse lungs. (A) Flow cytometry analysis to gate out total AEC2s (R2), and ZIP8 expression AEC2s (R3) from lung homologies of young and old mice. (B) Percentage of ZIP8+ cells (R3) within total AEC2s (n = 5–6, **P < 0.01). (C) Number of ZIP8+ cells recovered from young and old mouse lung (n = 5–6, **P < 0.01). (D) CFE of mouse AEC2s isolated from young and old mouse lungs (n = 6–7, **P < 0.01). (E and F) Flow cytometry analysis of ZIP8 expression in gated AEC2s cultured with medium only or medium containing 100 μM ZnSO4 (n = 6, ***P < 0.001). (G–J) 3D organoid culture of AEC2s isolated from lungs of 2.5-, 12-, 14-, and 18-month-old mice with and without 100 μM ZnSO4 treatment. (G) CFE (n = 3–4, *P < 0.05, ***P < 0.001, ****P < 0.0001 by ANOVA). (H–J) Expression of Slc39a8 (H), Sftpc (I), and Pdpn (J) in AEC2s derived from 3D-cultured organoids with and without ZnSO4 treatment by qPCR (n = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA). (K) Violin plots of gene expression in AEC2s from lungs of bleomycin-treated young and old mice. (L) IPA pathway analysis of AEC2s from young and old mice on day 4 after bleomycin injury. (M and N) CFE of AEC2s from uninjured 10- to 12-week-old young mice treated with SRT1720 (n = 3–6, **P < 0.01, ****P < 0.0001 by ANOVA) (M) and splitomicin (n = 5–6, ****P < 0.0001 by ANOVA) (N) at the indicated doses and DMSO control. (O) CFE of AEC2s from uninjured 20- to 24-month-old mice treated with SRT1720 at the indicated doses and DMSO control (n = 4, **P < 0.01, ****P < 0.0001 by ANOVA). Data are shown as mean ± SEM. Unpaired 2-tailed Student’s t test.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Flow Cytometry, Expressing, Isolation, Cell Culture, Derivative Assay, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 5. Targeted deletion of Slc39a8 decreased AEC2 renewal. (A) ZIP-expressing cells among gated AEC2s and (B) percentage of ZIP8+ cells within the total AEC2 population from uninjured Zip8AEC2 and control mice by flow cytometry (n = 8, ****P < 0.001). (C and D) Intracellular zinc levels of AEC2s (C) and percentage of zinc+ AEC2s within the total AEC2 population (D) from Zip8AEC2 (n = 4) and control mice (n = 8) by flow cytometry (**P < 0.01). (E) CFE of flow-sorted AEC2s from uninjured Zip8AEC2 and control mice with 3D organoid culture (n = 6 each, *P < 0.05). (F and G) Expression of Sirt1 (n = 4, *P < 0.05) (F) and Pdpn (n = 5, **P < 0.01) (G) in AEC2s derived from 3D-cultured organoids. (H–J) AEC2s from day 4 bleomycin-injured Zip8AEC2 and control mice. (H) Number of AEC2s recovered per lung (n = 5, ***P < 0.001). (I and J) CFE of AEC2s with 3D organoid culture (n = 5–7, **P < 0.01) (I) and colony size (n = 28–86, ****P < 0.0001) (J). (K and L) Ki-67 expression by flow cytometry (n = 6 each, *P < 0.05) (K) and Pdpn expression by qPCR (n = 5 each, ****P < 0.0001) (L) in AEC2s derived from 3D-cultured organoids. (M and N) Violin plots of gene expression in AEC2s with scRNA-Seq. (M) AEC2s from 2-month-old (Young) and 18- to 20-month-old (Old) C57BL/6 WT mice (n = 3). (N) AEC2s from 10- to 12-week-old (young) Zip8AEC2 mice and littermate controls 2 weeks after 4 doses of tamoxifen injection. Data are shown as mean ± SEM. Unpaired 2-tailed Student’s t test.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Expressing, Control, Flow Cytometry, Derivative Assay, Cell Culture, Gene Expression, Injection

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 7. Summary of the role of the ZIP8/SIRT1 axis in regulating alveolar progenitor cell renewal. In young and healthy AEC2s, sufficient ZIP8 ensures adequate levels of intracellular zinc, SIRT1 activity, and AEC2 renewal capacity. However, in old AEC2s and IPF AEC2s, severely downregulated ZIP8 results in intracellular zinc deficiency and defective SIRT1 activity, which impairs AEC2 renewal. In addition, enzymes regulating NAD+ synthesis were downregulated in IPF AEC2s, further exaggerating SIRT1 impairment. Therefore, the optimal combinations of zinc, NAD+, and SIRT1 activation may restore AEC2 integrity and mitigate fibrosis. MT, metallothionein.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Activity Assay, Activation Assay