Journal: Scientific Reports

Article Title: Marein from Coreopsis tinctoria Nutt. alleviates oxidative stress and lipid accumulation via SIRT1/Nrf2 signaling

doi: 10.1038/s41598-025-97964-7

Figure Lengend Snippet: Marein restored SIRT1/Nrf2 signaling in H 2 O 2 -induced HepG2 cells. HepG2 cells were exposed 500μM H 2 O 2 condition for 24h, and then administrated by 5 μM Marein for another 24h. ( a , b ) mRNA expression levels of SIRT1 ( a ) and Nrf2 ( b ) were assessed using RT-qPCR. ( c ) Western blot analysis of SIRT1 and Nrf2 protein levels. ( d ) Immunofluorescence staining of Nrf2 nuclear localization (scale bar = 25 μm). Data are represented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Besides, HepG2 cells was incubated with 5 μM Marein (≥ 96% purity, Shanghai Yuanye Bio-Technology Co., Ltd., China) for 24 h . For inhibitor experiments, cells were pre-treated with SIRT1 inhibitor EX-527 (100 nM, #A10377, AdooQ BioScience, Nanjing, China) for 2 h before Marein treatment.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Marein from Coreopsis tinctoria Nutt. alleviates oxidative stress and lipid accumulation via SIRT1/Nrf2 signaling

doi: 10.1038/s41598-025-97964-7

Figure Lengend Snippet: Inactivation of SIRT1/Nrf2 reversed the antioxidant ability of marein in H 2 O 2 -induced HepG2 cells. HepG2 cells were subjected to different groups: control, H 2 O 2 , H 2 O 2 + Marein, H 2 O 2 + Marein + EX-527 (SIRT1 inhibitor). ( a ) CCK-8 detection was used to examine cell viability. ( b ) LDH content was detected to examine oxidative stress injury. ( c – e ) MDA ( c ), SOD ( d ), and GSH-Px ( e ) levels were detected by ELISA. ( f ) DCFDA detected ROS level. Data are represented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Besides, HepG2 cells was incubated with 5 μM Marein (≥ 96% purity, Shanghai Yuanye Bio-Technology Co., Ltd., China) for 24 h . For inhibitor experiments, cells were pre-treated with SIRT1 inhibitor EX-527 (100 nM, #A10377, AdooQ BioScience, Nanjing, China) for 2 h before Marein treatment.

Techniques: Control, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Marein from Coreopsis tinctoria Nutt. alleviates oxidative stress and lipid accumulation via SIRT1/Nrf2 signaling

doi: 10.1038/s41598-025-97964-7

Figure Lengend Snippet: Inactivation of SIRT1/Nrf2 restrained protective role of marein in H 2 O 2 -triggered lipid accumulation. HepG2 cells were subjected to different groups: control, H 2 O 2 , H 2 O 2 + Marein, H 2 O 2 + Marein + EX-527 (SIRT1 inhibitor). ( a – d ) Determination of TC ( a ), TG ( b ), LDL-C ( c ), and HDL-C ( d ) levels by ELISA. ( e , f ) Detection of lipid metabolism related genes HMGCR ( e ) and LDLR ( f ) by RT-qPCR. ( g ) Western blot analysis of HMGCR and LDLR protein levels. Values were expressed as mean ± SD of three separate determinations. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Besides, HepG2 cells was incubated with 5 μM Marein (≥ 96% purity, Shanghai Yuanye Bio-Technology Co., Ltd., China) for 24 h . For inhibitor experiments, cells were pre-treated with SIRT1 inhibitor EX-527 (100 nM, #A10377, AdooQ BioScience, Nanjing, China) for 2 h before Marein treatment.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes

doi: 10.1371/journal.pone.0133448

Figure Lengend Snippet: (A) Chemical structures of the SIRT1 modulators EX527 (an inhibitor), SRTCX1002 (an activator) and SRTCZ1001 (a placebo). (B) HEK293 cells were treated with DMSO (blank control, I) or EX527 (II) for 48 hours or EX527 for 24 hours and then switched to SRTCX1002 (III) or SRTCZ1001 (IV) treatment for an additional 24 hours (left panel). The acetylation levels of p53 lysine-382, a known target of SIRT1 deacetylation, were examined by western blotting using a specific antibody. The p53 and SIRT1 protein levels were also determined by western blotting. β-tubulin was used as a loading control.

Article Snippet: SIRT1 inhibitor (EX527), SIRT1 activator (SRTCX1002), and placebo (SRTCZ1001) were provided by GlaxoSmithKline (GSK).

Techniques: Control, Western Blot

Journal: PLoS ONE

Article Title: SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes

doi: 10.1371/journal.pone.0133448

Figure Lengend Snippet: (A) Schematic illustration of the experimental procedure. Briefly, 3T3-L1 cells were differentiated into mature adipocytes for 6 days. Then, the mature adipocytes were treated with SIRT1 modulators as follows: (I) DMSO for 48 hours; (II) EX527 for 48 hours; (III) EX527 for 24 hours followed by a switch to SRTCX1002 for 24 hours; and (IV) EX527 for 24 hours followed by a switch to SRTCZ1001 for 24 hours. (B) SIRT1 protein levels were examined at the indicated times during 3T3-L1 adipogenesis by western blotting using an antibody against the C-terminal portion of SIRT1. Calnexin was used as a loading control. (C) Patterns of global protein acetylation in 3T3-L1 mature adipocytes after SIRT1 modulator treatments, as described in panel (A). The protein acetylation state was evaluated using a pan-acetyl-lysine antibody. The SIRT1 protein level was evaluated using an antibody against the C-terminal portion of SIRT1. Calnexin was used as a loading control.

Article Snippet: SIRT1 inhibitor (EX527), SIRT1 activator (SRTCX1002), and placebo (SRTCZ1001) were provided by GlaxoSmithKline (GSK).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes

doi: 10.1371/journal.pone.0133448

Figure Lengend Snippet: (A) Representative 2D-PAGE gel transfer blot stained with Coomassie blue and utilized in western blot analysis using a pan-acetyl-lysine antibody. (B) Western blot spots corresponding to ATP6V1B2 showed differential acetylation states upon SIRT1 modulator treatments, as described in . The spot intensities were quantified and are shown as a bar graph in the right panel. The value for the blank control was arbitrarily set as one. (C) The protein level of ATP6V1B2 was examined by western blot analyses after SIRT1 modulator treatment of 3T3-L1 mature adipocytes. Calnexin was used as a loading control.

Article Snippet: SIRT1 inhibitor (EX527), SIRT1 activator (SRTCX1002), and placebo (SRTCZ1001) were provided by GlaxoSmithKline (GSK).

Techniques: Staining, Western Blot, Control

Journal: PLoS ONE

Article Title: SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes

doi: 10.1371/journal.pone.0133448

Figure Lengend Snippet: (A) The sub-cellular localization of SIRT1 and ATP6V1B2 was determined by western blot analyses of fractionated mitochondrial (Mito), nuclear (Nuclei), and cytosolic (Cyto) samples from 3T3-L1 mature adipocytes. Markers of the mitochondrial (HK-1), nuclear (histone H2A) and cytosolic (β-tubulin) fractions were examined by western blotting to verify the purity of our preparations. Whole-cell lysate (WCL) was included as a positive control. (B) Sub-cellular localization of SIRT1 and ATP6V1B2 was examined by immunofluorescence analysis of 3T3-L1 mature adipocytes using a SIRT1 antibody and an ATP6V1B2 antibody, respectively. Nuclei were stained with DAPI. (C) Flag-tagged full-length SIRT1 (Flag-SIRT1) was expressed in HEK293 cells and immunoprecipitated by M2-agarose. The interaction between ATP6V1B2 and Flag-SIRT1 was examined by western blot analysis of ATP6V1B2 in the Flag-IP sample. The protein levels of SIRT1, Flag-SIRT1 and ATP6V1B2 were assessed by western blotting using the corresponding antibodies. β-tubulin was used as a loading control. The asterisk indicates a non-specific band detected by the Flag antibody.

Article Snippet: SIRT1 inhibitor (EX527), SIRT1 activator (SRTCX1002), and placebo (SRTCZ1001) were provided by GlaxoSmithKline (GSK).

Techniques: Western Blot, Positive Control, Immunofluorescence, Staining, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes

doi: 10.1371/journal.pone.0133448

Figure Lengend Snippet: (A) Acetylated p53 (Ac-p53) peptide was deacetylated by SIRT1 in vitro . The deacetylation level of the peptide was evaluated by measuring the fluorescent signal from free ammonia, a byproduct of the deacetylation reaction catalyzed by sirtuin enzymes. EX527, a specific SIRT1 inhibitor, significantly reduced the ability of SIRT1 to deacetylate the Ac-p53 peptide. (B) ATP6V1B2 protein purified from wheat germ is acetylated at lysine residues. The acetylation status of ATP6V1B2 was examined using a pan-acetyl-lysine antibody in the western blot analysis. Recombinant histone H3 (rH3) was used as a negative control for lysine acetylation. The protein levels were determined by Coomassie Brilliant Blue (CBB) staining. (C) ATP6V1B2 was deacetylated by SIRT1 in vitro . The data presented in this Fig are based on three independent experiments (mean ± SED) (** P < 0.01). (D) Endogenous ATP6V1B2 was immunoprecipitated by a specific antibody in HEK293 cells with or without over-expression of His/Myc-tagged full-length SIRT1 (SIRT1-His/Myc). The state of lysine acetylation of ATP6V1B2 was examined by western blot analysis using a pan-acetyl-lysine antibody in the IP sample. The protein levels of SIRT1, SIRT1-His/Myc and ATP6V1B2 were assessed by western blotting using the corresponding antibodies. β-tubulin was used as a loading control.

Article Snippet: SIRT1 inhibitor (EX527), SIRT1 activator (SRTCX1002), and placebo (SRTCZ1001) were provided by GlaxoSmithKline (GSK).

Techniques: In Vitro, Purification, Western Blot, Recombinant, Negative Control, Staining, Immunoprecipitation, Over Expression, Control

Journal: Lipids in Health and Disease

Article Title: CircLDLR acts as a sponge for miR-667-5p to regulate SIRT1 expression in non-alcoholic fatty liver disease

doi: 10.1186/s12944-022-01740-9

Figure Lengend Snippet: miR-667-5p can target SIRT1 to regulate the autophagy signaling pathway. A qRT-PCR detection of miR-667-5p level in Hepa1-6 cells. *** P < 0.001 vs. NC mimic group. B qRT-PCR detection of SIRT1 mRNA expression in Hepa1-6 cells. * P < 0.05 vs. NC mimic group. C SIRT1 levels in NAFLD mice were detected by western blot. *** P < 0.001 vs. NC mimic group. D The potential binding sequences between circLDLR and SIRT1 were predicted by TargetScan. E The binding association of miR-667-5p with SIRT1 was verified by determining the luciferase activity. * P < 0.05 vs. WT + NC mimic group. F and G The TG and TC contents in Hepa1-6 cells were measured via enzymatic method. * P < 0.05 vs. vector + NC mimic group; # P < 0.05 vs. vector + miR-667-5p mimic group. H SIRT1, p62, LC3, and mTOR expressions in Hepa1-6 cells were determined using western blot and relative quantification by densitometry. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vector + NC mimic group; # P < 0.05, ### P < 0.001 vs. SIRT1 OE + NC mimic group; & P < 0.05, &&& P < 0.001 vs. vector + miR-667-5p mimic group

Article Snippet: The luciferase reporter vectors circLDLR-Wt, circLDLR-Mut, SIRT1-Wt, and SIRT1-Mut were created via inserting wild-type (Wt) or mutant (Mut) sequences of circLDLR or SIRT1 3ʹUTR with the putative miR_668_5p binding sites into the pmirGLO plasmid (Promega, Madison, WI, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, Luciferase, Activity Assay, Plasmid Preparation

Journal: Lipids in Health and Disease

Article Title: CircLDLR acts as a sponge for miR-667-5p to regulate SIRT1 expression in non-alcoholic fatty liver disease

doi: 10.1186/s12944-022-01740-9

Figure Lengend Snippet: circLDLR regulates the SIRT1-autophagy signaling pathway to alleviate cells’ lipid accumulation. A The SIRT1 mRNA expression in Hepa1-6 cells. *** P < 0.001 vs. NC siRNA group. B The SIRT1 protein level in Hepa1-6 cells was determined using western blot. *** P < 0.001 vs. NC siRNA group. C ORO staining was determined to study the lipogenesis of si-SIRT1 in Hepa1-6 cells. ** P < 0.01 vs. NC group. D and E The TG and TC contents in Hepa1-6 cells were detected via an enzymatic method. * P < 0.05 vs. NC group; # P < 0.05 vs. vector group. F LC3 and p62 expressions in Hepa1-6 cells were determined through western blot and relative quantification. * P < 0.05, *** P < 0.001 vs. vector group; ## P < 0.01, ### P < 0.001 vs. circLDLR OE group; && P < 0.01, &&& P < 0.001 vs. vector + si-SIRT1 group

Article Snippet: The luciferase reporter vectors circLDLR-Wt, circLDLR-Mut, SIRT1-Wt, and SIRT1-Mut were created via inserting wild-type (Wt) or mutant (Mut) sequences of circLDLR or SIRT1 3ʹUTR with the putative miR_668_5p binding sites into the pmirGLO plasmid (Promega, Madison, WI, USA).

Techniques: Expressing, Western Blot, Staining, Plasmid Preparation