sirt1 inhibitor  (Santa Cruz Biotechnology)

Journal: Biomolecules

Article Title: Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

doi: 10.3390/biom12020323

Figure Lengend Snippet: PCR primer sets used in experiments.

Article Snippet: In separate experiments, when SIRT1 signaling’s involvement in VD3-mediated effects on BM-MSCs was evaluated, cells were treated for 5 days with VD3 (10 or 20 nM) in the presence or absence of the selective SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527, 5 μM) (Santa Cruz Biotechnologies); untreated cells served as controls.

Techniques: Sequencing

Journal: Biomolecules

Article Title: Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

doi: 10.3390/biom12020323

Figure Lengend Snippet: VD3 activates SIRT1-FoxO3 signaling pathways in BM-MSCs: BM-MSCs were treated with VD3 (10 nM or 20 nM) for 5 days under standard cultivation conditions. Protein expression of ( A ) SIRT1 and ( C ) FoxO3 following the treatment, as detected by indirect immunofluorescence staining. Secondary fluorescein isothiocyanate (FITC)-conjugated antibodies were used for labeling corresponding primary antibodies. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images from three independent experiments are shown (scale bars: 10 µm). ( B ) Relative expression levels for SIRT1 mRNA analyzed by qPCR were normalized to the Ct value of the housekeeping gene GAPDH. For calculations, the 2 −ΔΔ CT method was used. Results are presented as the mean ± SEM from at least two independent experiments. Statistically significant differences: *** p < 0.001 compared to untreated controls.

Article Snippet: In separate experiments, when SIRT1 signaling’s involvement in VD3-mediated effects on BM-MSCs was evaluated, cells were treated for 5 days with VD3 (10 or 20 nM) in the presence or absence of the selective SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527, 5 μM) (Santa Cruz Biotechnologies); untreated cells served as controls.

Techniques: Protein-Protein interactions, Expressing, Immunofluorescence, Staining, Labeling

Journal: Biomolecules

Article Title: Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

doi: 10.3390/biom12020323

Figure Lengend Snippet: VD3 treatment modulates proliferation and cell cycle progression of BM-MSCs, independently of SIRT1 signaling. BM-MSCs were treated for 5 days with VD3 (10 nM or 20 nM), EX-527 (5 µM), or both, under standard cultivation conditions. ( A ) Metabolic activity estimated by MTT test. Results are presented as the mean ± SEM for at least three independent experiments. Statistically significant differences: ** p < 0.01; *** p < 0.001 in comparison to untreated controls (Ctrl). ( B ) Expression of the proliferation-associated protein Ki67 in BM-MSCs, as detected by flow cytometry. Representative histograms illustrating the percentage of Ki67-positive cells (pink peaks) versus unstained cells as negative controls (blue peaks). ( C ) Ki67 expression detected by indirect immunofluorescence staining of BM-MSCs. Primary antibodies were labelled with corresponding fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, while DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images from three independent experiments conducted in duplicate are shown (scale bars: 10 µm). ( D ) Cell cycle distribution as determined by flow cytometric analysis after propidium iodide (PI) staining. Results from at least three independent experiments are presented in the graph as means ± SEM. Statistically significant differences: * p < 0.05; ** p < 0.01 in comparison to untreated controls.

Article Snippet: In separate experiments, when SIRT1 signaling’s involvement in VD3-mediated effects on BM-MSCs was evaluated, cells were treated for 5 days with VD3 (10 or 20 nM) in the presence or absence of the selective SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527, 5 μM) (Santa Cruz Biotechnologies); untreated cells served as controls.

Techniques: Activity Assay, Comparison, Expressing, Flow Cytometry, Immunofluorescence, Staining

Journal: Biomolecules

Article Title: Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

doi: 10.3390/biom12020323

Figure Lengend Snippet: VD3 treatment modulates the expression of pluripotency-associated markers in BM-MSCs: involvement of SIRT1 signaling: BM-MSCs were treated for 5 days with VD3 (10 nM or 20 nM), EX-527 (5 µM), or both. Immunocytochemical detection of pluripotency-related transcription factors: ( A ) NANOG, ( B ) Oct4, and ( C ) SOX2. Primary antibodies were labeled with appropriate FITC-conjugated secondary antibodies, while cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images from three independent experiments conducted in triplicate are shown (scale bars: 10 µm). Graphical presentation of pluripotency markers’ mRNA expression as analyzed by qPCR: relative gene expression levels for ( D ) NANOG, ( E ) Oct4, and ( F ) SOX2, normalized to the Ct value of the housekeeping gene GAPDH. Calculations were performed by applying the 2 −ΔΔ CT method, and results were presented as the mean ± SEM from at least two independent experiments. Statistically significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated controls; # p < 0.05 compared to corresponding VD3 treatment without EX-527.

Article Snippet: In separate experiments, when SIRT1 signaling’s involvement in VD3-mediated effects on BM-MSCs was evaluated, cells were treated for 5 days with VD3 (10 or 20 nM) in the presence or absence of the selective SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527, 5 μM) (Santa Cruz Biotechnologies); untreated cells served as controls.

Techniques: Expressing, Labeling, Staining, Gene Expression

Journal: Biomolecules

Article Title: Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

doi: 10.3390/biom12020323

Figure Lengend Snippet: Stimulated osteogenic differentiation of VD3-pretreated BM-MSCs: involvement of SIRT1 signaling: BM-MSCs were pretreated for 5 days with VD3 (10 nM or 20 nM), EX-527 (5 µM), or both, in GM. Afterwards, the cells were cultured in OM for the appropriate period to induce osteogenesis. ( A ) ALP activity detected after 7 days of cultivation in OM by histochemical staining. ( B ) Calcium depositions stained with Alizarin Red after 21 days. Representative images are shown (scale bars: 50 µm). Graphs represent quantitative analysis of ( A ) ALP activity and (B) mineralization level for at least three independent experiments. Results are normalized to values for untreated controls and presented as means ± SEM. Statistically significant differences: ** p < 0.01; *** p < 0.001 compared to untreated OM controls; # p < 0.05, ## p < 0.01 compared to corresponding pretreatment with VD3 in the absence of EX-527. ( C ) For quantitative real-time PCR analysis of the ALP, RUNX2, and OCN mRNA expression, cells were grown for 5 days in GM in the presence of VD3 (10 nM or 20 nM), EX-527 (5 µM), or both. Relative expression levels for ALP, RUNX2, and OCN genes were normalized to the Ct value of the housekeeping gene GAPDH. Calculations by the 2 −ΔΔ CT method are presented as the mean ± SEM from at least two independent experiments. Statistically significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated controls; # p < 0.05 compared to corresponding VD3 treatment without EX-527.

Article Snippet: In separate experiments, when SIRT1 signaling’s involvement in VD3-mediated effects on BM-MSCs was evaluated, cells were treated for 5 days with VD3 (10 or 20 nM) in the presence or absence of the selective SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527, 5 μM) (Santa Cruz Biotechnologies); untreated cells served as controls.

Techniques: Cell Culture, Activity Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Biomolecules

Article Title: Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

doi: 10.3390/biom12020323

Figure Lengend Snippet: VD3 treatment reduces β-galactosidase expression in BM-MSCs: the role of SIRT1 signaling: Identification of β-galactosidase-positive cells was conducted following 5-day treatment with VD3 (10 nM or 20 nM), EX-527 (5 μM), or both, in GM. Representative images of β-galactosidase-positive cells ( left ); scale bars: 50 μm. The percentage of β-galactosidase-positive cells was enumerated per visual field and presented as the mean ± SEM of three independent experiments ( right ). Statistically significant differences: * p < 0.05, ** p < 0.01 compared to untreated controls (Ø); ## p < 0.01 compared to corresponding VD3 treatment without EX-527.

Article Snippet: In separate experiments, when SIRT1 signaling’s involvement in VD3-mediated effects on BM-MSCs was evaluated, cells were treated for 5 days with VD3 (10 or 20 nM) in the presence or absence of the selective SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527, 5 μM) (Santa Cruz Biotechnologies); untreated cells served as controls.

Techniques: Expressing

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: Manipulation of SIRT1 activity affected excitatory synaptic transmission in dentate gyrus from hippocampus slices of control mice. SIRT1 inhibition with SIRT1 inhibitor IV (1 µM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs). a Voltage-clamp recording of sEPSCs recorded at −70 mV from the same cell in DMSO and SIRT1 inhibitor IV. b Summary graph showing sEPSC frequency from all cells in DMSO and SIRT1 inhibitor IV ( n = 10). SIRT1 activator 3 (50μM) reduced the frequency of sEPSCs compared to DMSO. c Voltage-clamp recording of sEPSCs recorded at −70 mV in the same cell in control (DMSO) and SIRT1 activator 3. d Summary plot showing sEPSC frequency in DMSO and SIRT1 activator 3 ( n = 11). e Voltage-clamp recording of miniature EPSCs recorded at −70 mV in the same cell in TTX and SIRT1 inhibitor. f Summary plot showing that SIRT1 inhibition ( n = 9) increased the frequency but not mEPSCs (mEPSCs) compared to TTX. g Voltage-clamp recording of mEPSCs recorded at −70 mV in the same cell in control (DMSO) and SIRT1 activator 3. h Summary graph showing frequency in DMSO and SIRT1 activator 3 ( n = 11). SIRT1 activator reduced the frequency of mEPSCs (* P < 0.05, ** P < 0.01; paired t -test; mean ± SEM shown). Scale bar = 20 pA and 5 s

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Activity Assay, Transmission Assay, Inhibition

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: Manipulation of SIRT1 activity affected spike width and fast afterhyperpolarization amplitude in the dentate gyrus granule cells in slices from control mice. SIRT1 inhibition increased spike width and decreased fast afterhyperpolarization amplitude. a Example of action potential recorded in control (black) and in SIRT1 inhibitor IV (blue). b Summary graphs showing that SIRT1 inhibitor increased spike-width and decreased fast afterhyperpolarization amplitude ( c ) in dentate granule cells ( n = 12). SIRT1 activation had no effect on spike width but decreased the fast afterhyperpolarization amplitude. d Example of action potential recorded in control (black) and SIRT1 activator (red). e SIRT1 activator had no effect on spike width, but significantly decreased fast afterhyperpolarization amplitude ( f , n = 9) (* P < 0.05, ** P < 0.01; n.s., not significant; paired t -test; mean ± SEM shown). Scale bar = 10 mV and 2 ms

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Activity Assay, Inhibition, Activation Assay

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: Paxilline pre-incubation blocked the effects of SIRT1 inhibition on spike width and fast afterhyperpolarization amplitude. a Preincubation of slices with paxilline to block BK channels blocked the effects of SIRT1 inhibition of spike width and fast afterhyperpolarization amplitude ( n = 9). The overlapped spike trace before (black) and after (blue) application of SIRT1 inhibitor IV in cells pre-incubated with paxilline. Scale bar = 10 mV and 2 ms. b Summary graph showing the effect of SIRT1 inhibitor on spike width and fast afterhyperpolarization amplitude ( c ) in the presence of paxilline. d Voltage-clamp recording of sEPSCs in the presence of paxilline and paxilline + SIRT1 inhibitor. Scale bar = 10 pA and 2 s. e Summary graph showing preincubation with paxilline blocked the effect of SIRT1 inhibition on sEPSC frequency ( n = 9) (n.s., not significant; paired t -test, mean ± SEM shown)

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Incubation, Inhibition, Blocking Assay

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: The BK α subunit interacts with SIRT1 and is a target of SIRT1. a Immunoprecipitation (IP) with SIRT1 and immunoblot with BK α antibody and IP with BK α and IB with SIRT1 shows that SIRT1 and BK α interact. b IP with BK α subunit shows that BK β subunits interacted with the α subunit but not with SIRT1. c Co-IP with the BKα subunit and anti-acetylated lysine antibody shows that BK α is acetylated. d SIRT1 activation and inhibition in slices and subsequent IP with the BK subunit and immunoblot with the anti-acetylated lysine antibody demonstrated that the BK α subunit is acetylated in hippocampus slices and the acetylation level is regulated by SIRT1 activity. e summary bar graph of BK α acetylation showing the percent change from control (DMSO) in the SIRT1 activation and inhibition experiments. SIRT1 inhibition significantly increased BK channel acetylation (** P < 0.01, n = 5; post-hoc Dunnett’s multiple comparisons test, mean ± SEM shown). The original blots for each figure can be found in supplementary figure

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Activity Assay

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: SIRT1 manipulation affected BKα surface expression. a BS3 crosslinking of surface proteins shows that SIRT1 activation decreased membrane BKα distribution (surface band). The original blots can be found in supplementary figure . b Summary graph showing SIRT1 activation significantly decreased BKα surface expression (Student’s t -test, n = 6; * P = 0.02)

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Expressing, Activation Assay

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: Modulation of SIRT1 activity had no effect on excitatory synaptic transmission in dentate gyrus from hippocampus slices of CVS-treated mice. SIRT1 inhibition with SIRT1 inhibitor IV (1 µM) had no effect on sEPSCs in slices from CVS-treated mice. a Voltage-clamp recording of sEPSCs recorded at −70 mV from the same cell in vehicle (DMSO) and SIRT1 inhibitor IV. b Summary plot showing sEPSC frequency from all cells in DMSO and SIRT1 inhibitor IV ( n = 9). Activation of SIRT1 had no effect on the frequency of sEPSCs in slices from CVS-treated mice. c Voltage-clamp recording of sEPSCs recorded at −70 mV. d Summary plot showing frequency in DMSO and SIRT1 activator ( n = 9). e Voltage-clamp recording of mESPCs recorded at −70 mV in TTX and SIRT1 inhibitor IV. f SIRT1 inhibition ( n = 10) had no significant effect on the frequency of mEPSCs compared to vehicle (DMSO) in slices from CVS-treated mice. g Voltage-clamp recording of mESPCs recorded at −70 mV in TTX and SIRT1 activator 3. h SIRT1 activation ( n = 10) had no effect on the frequency of mEPSCs in slices from CVS-treated mice (n.s., not significant, paired t -test, mean ± SEM shown). Scale bar = 20 pA and 5 s

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Activity Assay, Transmission Assay, Inhibition, Activation Assay

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: SIRT1 manipulation had no effect on spike width and fast afterhyperpolarization amplitude in the dentate gyrus granule cells in slices from CVS-treated mice. SIRT1 inhibition had no effect on spike width or fast afterhyperpolarization amplitude. a Example of action potential recorded in control (black) and in SIRT1 inhibitor (blue). b Summary graph showing that SIRT1 inhibitor had no effect on spike width or fast afterhyperpolarization amplitude ( c n = 9) in slices from CVS-treated mice. d Example of action potential recorded in control (black) and in SIRT1 activator (red). e Summary graph showing that SIRT1 activation had no effect on spike width or fast afterhyperpolarization amplitude ( f ) in slices from CVS-treated mice ( n = 9) (n.s.,not significant, paired t -test, mea n ± SEM shown). Scale bar = 10 mV and 2 ms

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Inhibition, Activation Assay

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: Acute stress increased SIRT1 activation in the dentate gyrus. Animals were exposed to trimethylthiazoline for 30 min as a single acute stressor. a Example Western blot showing the levels of acetylated p53 and total p53 in control ( n = 10) and after acute stress ( n = 10). b The levels of acetylated p53 relative to total p53 measured by immunoblot were significantly decreased by acute stress in the dentate gyrus (**, p < 0.01). The original blots can be found in supplementary figure

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Activation Assay, Western Blot

Journal: Communications Biology

Article Title: BK channel deacetylation by SIRT1 in dentate gyrus regulates anxiety and response to stress

doi: 10.1038/s42003-018-0088-5

Figure Lengend Snippet: Direct infusion of SIRT1 inhibitor IV and SIRT1 activator 3 into the dentate gyrus rapidly affected anxiety behavior in open field test, and these effects were occluded by BK channel blockade. a Schematic diagram showing injection points (black circles) using coordinates from Paxinos and Franklin atlas to demonstrate placement of infusions into the dentate gyrus. b Example heat map of time spent in the field from the same mouse when infused with saline with DMSO, SIRT1 activator or SIRT1 inhibitor (top) and in the presence of paxilline (bottom). c Summary plot of distance traveled in the open field in animals infused with saline or SIRT1 activator or inhibitor. d Summary plot of percent time spent exploring in the center of the field. The results indicated SIRT1 activity significantly affected the time spent in the center (one-way repeated measures ANOVA, ** P < 0.01). SIRT1 activator 3 perfusion increased the time spent in the center (paired t -test, #, P = 0.07) and SIRT1 inhibitor IV perfusion significantly decreased the time spent in the center of the arena (paired t -test, * P < 0.05). e Summary plot of distance traveled in the open field in with SIRT1 activation and inhibition in the presence of paxilline to block BK channels. f Summary plot of time spent exploring in the center of the open field. Paxilline occluded the effects of SIRT1 activation and inhibition

Article Snippet: The main SIRT1 inhibitor and activator drugs were made as 1000× stock solution in DMSO stored aliquoted at −20 ℃, and bath applied at a concentration demonstrated before , prior to bath application: SIRT1 inhibitor IV (Calbiochem Cat #566325): 1 µM; SIRT1 activator 3 (Santa Cruz Biotechology, Inc. sc-222315): 50 µM.

Techniques: Injection, Activity Assay, Activation Assay, Inhibition, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells

doi: 10.3390/ijms18040852

Figure Lengend Snippet: SIRT1 activity and overexpression is involved in the fisetin-mediated cytoprotective effect. ( a , b ) Fisetin reverses Tm-mediated SIRT1 downregulation. PC12 cells were treated with fisetin (5–20 µM) 30 min prior to Tm (1 µg/mL) treatment for 16 h at 37 °C. Cell lysates were prepared and the levels of SIRT1 and α-tubulin were analyzed by immunoblotting. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with control. ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group; ( c ) Effects of inhibition of SIRT1 activity on cell viability. PC12 cells were pretreated for 30 min with 15 µM sirtinol and 10 or 20 µM fisetin was then added 30 min prior to Tm (1 µg/mL) exposure for 16 h. MTT was used to analyze the cell viability. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. ## p < 0.01 represents significant differences compared with the respective no inhibitor group.

Article Snippet: Sirtinol, a specific inhibitor of SIRT1 and SIRT2, was from Santa Cruz (Dallas, TX, USA).

Techniques: Activity Assay, Over Expression, Western Blot, Control, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells

doi: 10.3390/ijms18040852

Figure Lengend Snippet: Primary antibodies used in Western blotting.

Article Snippet: Sirtinol, a specific inhibitor of SIRT1 and SIRT2, was from Santa Cruz (Dallas, TX, USA).

Techniques: Western Blot