Journal: iScience

Article Title: Mitochondrial hyper-acetylation induced by an engineered acetyltransferase promotes cellular senescence

doi: 10.1016/j.isci.2025.113233

Figure Lengend Snippet: Global mitochondrial acetylation controlled by eMAT and SIRT3 (A) A schematic for quantitative LC-MS/MS. Mitochondria fractions from parental, eMAT treated with 100 ng/mL Dox, eMAT+SIRT3 treated with 100 ng/mL Dox were isolated and treated with trypsin. The tryptic peptides were immunoprecipitated with anti-AcK antibodies, and captured with Protein-A/G agarose beads. The beads-bound peptides were eluted and analyzed with LC-MS/MS with a label-free quantitative method (Proteome Discoverer Ver.3.1). (B) Venn diagram of identified AcK-containing peptides. (C) Violin plot of the AcK peptides. Friedman Test: p = 4.295 × 10 −5 . (D) Comparison of acetylation between eMAT and control sample. Fold change of acetylated peptides (total 1240 peptides) was calculated as log2([peptide abundance of eMAT]/[peptide abundance of control]). (E) Ven diagram of the eMAT targets. Among 725 identified substrates with log2FC > 1, 74.3% were known proteins. (F) Consensus motif analysis of acetylation sites with WebLogo (Ver.3). GO analysis of eMAT targets with DAVID (Ver.2021): (G) Cellular compartment, (H) Biological process. (I) Comparison of acetylation between eMAT+SIRT3 and eMAT. Fold changes were calculated as log2([peptide abundance of eMAT+SIRT3]/[peptide abundance of eMAT]). (J) Ven diagram of the eMAT and SIRT3 targets. (K) Consensus motif analysis of deacetylation sites by SIRT3. GO analysis of eMAT and SIRT3 targets: (L) Cellular compartment, (M) Biological process.

Article Snippet: Acetylated Lysine (Ac-K2-100) MultiMab Rabbit mAb mix , Cell Signaling Technology , Cat#9814; RRID:AB_10544700.

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Immunoprecipitation, Comparison, Control

Journal: iScience

Article Title: Mitochondrial hyper-acetylation induced by an engineered acetyltransferase promotes cellular senescence

doi: 10.1016/j.isci.2025.113233

Figure Lengend Snippet: eMAT acetylates multiple mitochondrial metabolic enzymes and SOD2 (A) Validated metabolic enzymes acetylated by eMAT: Orange; eMAT-dependent acetylation, magenta; eMAT-dependent acetylation and SIRT3-dependent deacetylation. FLAG-tagged proteins were expressed in Dox-inducible eMAT cells (B) or eMAT+Vec and eMAT+SIRT3 cells (C), treated with 10 ng/mL Dox for 24 h. Immunoprecipitated proteins were blotted with anti-AcK antibodies or anti-FLAG antibodies. Expression of eMAT (anti-V5) and SIRT3 (anti-HA) was confirmed with immunoblot of total cell lysates. LC-MS/MS analysis of FLAG-tagged (D) ACO2, (E) DLST, (F) SDHA, (G) SUCLG1 in control (eMAT-V5+vec cells without Dox treatment), eMAT (eMAT-V5+vec cells treated with 10 ng/mL Dox for 24 h), eMAT+SIRT3 (eMAT-V5+SIRT3 treated with 10 ng/mL Dox for 24 h) cells. Normalized acetylation (%) was calculated based on the intensity of the acetylated peptide, normalized by the sum of the intensities of the corresponding unacetylated and acetylated peptides. Blue; common acetylation site whose acetylation was detected both in the control and eMAT. (H) Schematic of eMAT mediated acetylation of metabolic enzymes and SOD2. (I) Representative immunoblot of eMAT-dependent acetylation of SOD2. (J) Quantitation of SOD2 acetylation. n = 5; mean ± SEM. Tukey’s HSD test: p † < 0.1, p ∗ < 0.05. (K) LC-MS/MS analysis of FLAG-tagged SOD2 in control, eMAT, eMAT+SIRT3 cells. (L) Venn diagram showing the number of acetylation sites identified in control and eMAT conditions across the five substrates.

Article Snippet: Acetylated Lysine (Ac-K2-100) MultiMab Rabbit mAb mix , Cell Signaling Technology , Cat#9814; RRID:AB_10544700.

Techniques: Immunoprecipitation, Expressing, Western Blot, Liquid Chromatography with Mass Spectroscopy, Control, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells

doi: 10.3390/ijms25084147

Figure Lengend Snippet: Viability of BME-UV1 bovine mammary epithelial cells treated for 24 h with different concentrations of chemerin, leptin, or adiponectin. Graphs present cell viability measured using the MTT assay. The viability of untreated control cells (0) was designated as 100%. Results are presented as means ± standard deviation of three independent experiments.

Article Snippet: Chemerin (cat.no: 2325-CM-025), leptin (cat.no: 498-OB-01M), adiponectin (cat.no: 5095-AC-050), and prolactin (cat. no: 682-PL-050) were purchased from R&D systems (Minneapolis, MN, USA).

Techniques: MTT Assay, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells

doi: 10.3390/ijms25084147

Figure Lengend Snippet: Expression of adipokine receptors in BME-UV1 bovine mammary epithelial cells. ( A , B ) Expression of adiponectin receptors ( ADIPOR1 and ADIPOR2 ); ( C , D ) expression of chemerin receptors ( CMLKR1 and GPR1 ). The relative mRNA expression of the analyzed genes was normalized to the mean expression of the histone reference gene. The expression of each analyzed gene in cells cultured in control medium (Ctrl.) was given as 1. Treatment with adiponectin at concentrations of 10 ng/mL or 500 ng/mL is marked as A10 and A500, respectively. Treatment with chemerin at concentrations of 10 ng/mL or 100 ng/mL is marked as Ch10 and Ch100, respectively. Results are presented as means ± standard deviation of three independent experiments performed in duplicate. Values that differed significantly from control are marked as ** ( p < 0.01) or *** ( p < 0.001).

Article Snippet: Chemerin (cat.no: 2325-CM-025), leptin (cat.no: 498-OB-01M), adiponectin (cat.no: 5095-AC-050), and prolactin (cat. no: 682-PL-050) were purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells

doi: 10.3390/ijms25084147

Figure Lengend Snippet: Evaluation of apoptosis in BME-UV1 bovine mammary epithelial cells treated with chemerin (Ch10 = 10 ng/mL, Ch100 = 100 ng/mL), leptin (L10 = 10 ng/mL, L100 = 100 ng/mL), or adiponectin (A10 = 10 ng/mL, A500 = 500 ng/mL) for 24 h. ( A ) Representative dot-plots of Annexin V/PI double staining in untreated control cells; ( B ) graph showing percentage of apoptotic cells (sum of Annexin V pos /PI neg and Annexin V pos /Pi pos cells) in control and adipokine-treated cells; ( C ) representative images of Western blot (WB) analysis of apoptotic markers cleaved caspase 3 and bax, where gapdh was used as a reference protein; ( D , E ) graphs showing the results of the densitometric analysis of WB images; the integrated optical density (IOD) of cleaved caspase 3 and bax bands was normalized to the IOD of gapdh bands. Results are presented as means ± standard deviation of three or four independent experiments.

Article Snippet: Chemerin (cat.no: 2325-CM-025), leptin (cat.no: 498-OB-01M), adiponectin (cat.no: 5095-AC-050), and prolactin (cat. no: 682-PL-050) were purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Double Staining, Control, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells

doi: 10.3390/ijms25084147

Figure Lengend Snippet: Concentration of αS1-casein in BME-UV1 cells ( A ) and media ( B ) collected after 24 h of culture. Cell were treated with chemerin (Ch10 = 10 ng/mL, Ch100 = 100 ng/mL), leptin (L10 = 10 ng/mL, L100 = 100 ng/mL) or adiponectin (A10 = 10 ng/mL, A500 = 500 ng/mL). Untreated cells were used as a negative control, whereas cells exposed for 24 h to the lactogenic hormone prolactin (PRL, 1 µg/mL) were used as a positive control. Results are presented as means ± standard deviation of three independent experiments. Means followed by a common letter are not significantly different according to one-way ANOVA with Tukey’s multiple comparison post-test, at the 5% level of significance ( p < 0.05).

Article Snippet: Chemerin (cat.no: 2325-CM-025), leptin (cat.no: 498-OB-01M), adiponectin (cat.no: 5095-AC-050), and prolactin (cat. no: 682-PL-050) were purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Concentration Assay, Negative Control, Positive Control, Standard Deviation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells

doi: 10.3390/ijms25084147

Figure Lengend Snippet: Panels of confocal micrographs presenting the immunofluorescence staining of BME-UV1 cells cultured on Matrigel for 11 days in control growth medium (Ctrl.) or medium supplemented with chemerin (Ch10 = 10 ng/mL, Ch100 = 100 ng/mL), leptin (L10 = 10 ng/mL, L100 = 100 ng/mL), or adiponectin (A10 = 10 ng/mL, A500 = 500 ng/mL). Cells were stained with primary antibodies against β1-integrin and secondary antibodies conjugated with Alexa Fluor 488 dye (green fluorescence); Alexa Fluor 594 phalloidin, detecting F-actin (red fluorescence), and nuclei counterstained with Hoechst 33342 (blue fluorescence). Images were taken at 600× magnification and are representative for three independent experiments. Scale bar: 20 μm.

Article Snippet: Chemerin (cat.no: 2325-CM-025), leptin (cat.no: 498-OB-01M), adiponectin (cat.no: 5095-AC-050), and prolactin (cat. no: 682-PL-050) were purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Staining, Cell Culture, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells

doi: 10.3390/ijms25084147

Figure Lengend Snippet: Diameters of 3D mammospheres formed by BME-UV1 cells cultured on Matrigel for 11 days. Cells were cultured in control growth medium (Ctrl.) or medium supplemented with chemerin (Ch10 = 10 ng/mL, Ch100 = 100 ng/mL), leptin (L10 = 10 ng/mL, L100 = 100 ng/mL), or adiponectin (A10 = 10 ng/mL, A500 = 500 ng/mL). The diameters of cross sections of the spheroids were measured using ImageJ software ( https://ij.imjoy.io accessed on 2 April 2024). Results are presented as means ± standard deviation of at least 14 spheroids per experimental condition photographed using a confocal microscope equipped with a digital camera.

Article Snippet: Chemerin (cat.no: 2325-CM-025), leptin (cat.no: 498-OB-01M), adiponectin (cat.no: 5095-AC-050), and prolactin (cat. no: 682-PL-050) were purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Software, Standard Deviation, Microscopy