Journal: Cells

Article Title: SMYD3 Modulates AMPK-mTOR Signaling Balance in Cancer Cell Response to DNA Damage

doi: 10.3390/cells12222644

Figure Lengend Snippet: Effects of NCS on the AMPK/mTOR pathways. ( a ) Immunoblotting showing the levels of proteins involved in the AMPK and mTOR pathways in HCT-116, MDA-MB-231, and AGS cells treated for 24 h with NCS (5 nM). ( b ) Immunoblotting showing the levels of proteins involved in the AMPK and mTOR pathways in SMYD3-KO cells (HCT-116 and MDA-MB-231) treated for 24 h with NCS (5 nM). H2AX phosphorylation (γH2AX) was analyzed as a control of the induced-DNA damage, VINCULIN was used as a loading control.

Article Snippet: Primary antibodies used: anti-Acetyl-CoA Carboxylase (#3676, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Acetyl-CoA Carboxylase (Ser79) (#3661, Cell Signaling Technology, Danvers, MA, USA), anti-AMPK-α (#2532, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-AMPK-α (Thr172) (#2531, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Histone H2A.X (Ser139) (#9718, Cell Signaling Technology, Danvers, MA, USA), anti-p70 S6 Kinase (#34475, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-p70 S6 Kinase (Thr389) (#9234, Cell Signaling Technology, Danvers, MA, USA), anti-RAPTOR (#2280, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-RAPTOR (Thr792) (#89146, Cell Signaling Technology, Danvers, MA, USA), anti-S6 Ribosomal Protein (#2217, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-S6 Ribosomal Protein (Ser240/244) (#5364, Cell Signaling Technology, Danvers, MA, USA), anti-VINCULIN (#13901, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Gut microbiota-dependent increase in phenylacetic acid induces endothelial cell senescence during aging

doi: 10.1101/2023.11.17.567594

Figure Lengend Snippet: a, Schematic diagram of the experimental setting: μμProliferating ECs (PEC) at p4-5 were treated with PAA (10 μM) for 72 h and then subjected to senescence hallmarks profiling. b, SA-β-gal staining shows an increase in cellular senescence in PECs treated with PAA at the magnitude seen in replicative senescent ECs (SEC) at p15-17. Right, quantitative plots are shown for SA-β-gal-positive cells (%) (n=6). c, qPCR shows that PAA treatment resulted in increased expression of CDK inhibitors p16 INK4a , p19 INK4d , and p21 WAF1/Cip1 in PECs. d, Immunoblots (top) and γ-H2A.X immunostaining (bottom) represent DDR in PAA-exposed PECs compared to vehicle-treated PECs (n=6). e, qPCR demonstrates that SASP genes IL1α, IL-1β, IL-6, Tnfa, and VCAM1 are significantly upregulated in PAA-treated PECs (n=6). f, VCAM1 immunostaining shows its marked overexpression in PAA-induced senescent ECs (n=6). Data from in vitro cellular experiments represent triplicated biologically independent experiments. Scale bar, 20 μm. Error bars represent SD ( b-e ). P values were calculated using one-way ANOVA followed by Tukey’s post hoc test ( b-e ). (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns, not significant). Source data are provided as a Source Data file.

Article Snippet: Antibodies against phospho-CaMKII Thr286 (1:1000; 12716), total-CaMKII (1: 1000; 4436), phospho-HDAC4 Ser632 (1:1000; 3424), total-HDAC4 (1:1000; 7628), H3 (1:1000; 9715), phospho-eNOS Ser1177 (1:1000; 9517), phospho-eNOS Thr495 (1:1000; 9574), total-eNOS (1: 1000; 32027), and phospho-Histone H2A.X Ser139 (1:1000; 80312) were purchased from Cell Signaling Technology; antibodies against VCAM-1 (1:1000; MA5-31965), IL6 (1:1000; M-620), NADPH oxidase 2 (NOX2) (1:1000; PA5-76034), and VE Cadherin (1:100; PA5-19612) were obtained from Invitrogen; antibody against CD31 (1:100; 14-0311-82) was purchased from eBioscience; antibody against Histone H3ac (Pan-Acetyl) (1:500; sc-518011) was obtained from Santa Cruz Biotechnology; antibody against p16 INK4A (1:100; ZRB1437) was purchased from Merck Millipore; antibody against phospho-HDAC4 Ser632 (1:100; ab39408-1001) and conjugated secondary antibodies against Alexa Fluor ® 488 (1:2000; ab150157) and Alexa Fluor ® 647 (1:2000; ab150075) were obtained from abcam; antibodies against goat anti-rabbit IgG-HRP (1:2000; 4030-05) and goat anti-mouse IgG-HRP (1:2000; 1036-05) were purchased from Southern Biotechnology.

Techniques: Staining, Expressing, Western Blot, Immunostaining, Over Expression, In Vitro

Journal: bioRxiv

Article Title: Gut microbiota-dependent increase in phenylacetic acid induces endothelial cell senescence during aging

doi: 10.1101/2023.11.17.567594

Figure Lengend Snippet: a, Fecal samples from old and young mice were collected for quantification of acetate by HPLC-RI. Bar chart represents significantly lower levels of acetate in feces of old mice compared to young mice (n=6). b, Schematic diagram of the experimental setting: PECs were co-treated with PAA (10 μM) and sodium acetate (3 μM) for 72 h and then analyzed for senescence hallmarks. c, Representative images of SA-β-gal staining (left) and bar chart (right) demonstrate that sodium acetate reduces cellular senescence in PAA-exposed PECs, represented by SA-β-gal-positive cells (%) (n=6). d, Representative images of γ-H2A.X foci demonstrate a marked reduction in DDR in PECs co-treated with PAA and sodium acetate compared to PECs exposed to PAA alone (n=6). e-h, qPCR analysis reveals that sodium acetate downregulates PAA-induced CDK inhibitors, p16 INK4a ( e ) and p19 INK4d ( f ), and SASP genes, IL-1β ( g ) and IL-6 ( h ), in PECs (n=6). i, Immunoblots show that sodium acetate regulates IL6-stimulated CaMKII-HDAC4 phosphorylation and the subsequent VCAM1 expression and eNOS phosphorylation at specific sites Ser1177 and Thr495 in PAA-exposed PECs (n=6). j, VCAM1 immunofluorescence represents that sodium acetate reduces its overexpression stimulated by PAA in PECs (n=6). k, Model for the proposed role of sodium acetate in restoration of eNOS signaling pathway in PAA-exposed PECs. We propose that acetate-mediated downregulation of IL6 is accompanied by reduced CaMKII-HDAC4 phosphorylation that localizes HDAC4 in the cell nucleus and subsequently dampens VCAM1 overexpression and restores eNOS signaling. l, CellRox green fluorescence staining demonstrates acetate-mediated reduction in intracellular ROS generation in PECs in the presence of PAA. m, Bioenergetics analysis by Seahorse metabolic analyzer reveals mitochondrial OCR in PAA- versus acetate+PAA-treated PECs. Bar charts show that co-incubation of PECs with sodium acetate and PAA restores mitochondrial function, represented by significant increases in basal and maximal OCR, spare reserve, and ATP biosynthesis as opposed to PAA-exposed PECs (n = 6). n-p, Confocal micrographs depict markedly positive effects of sodium acetate to restore endothelial angiogenic capcity, represented by cell migration ( n ), tube formation ( o ), and aortic endothelial sprouting ( p ). Quantitative plots represent significantly higher PEC migrated area ratio (n), the number of tubes formed (o), and the number of endothelial sprouts from aortic rings in the acetate+PAA-treated group compared to PAA-exposed group. Scale bars, 20, 100, and 200 μm. Error bars represent SD ( a,c,e-I,n-p ) or SEM ( m ). Data represent triplicated biologically independent experiments. P values were calculated using a two-tailed unpaired Student’s t -test. (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001). Source data are provided as a Source Data file.

Article Snippet: Antibodies against phospho-CaMKII Thr286 (1:1000; 12716), total-CaMKII (1: 1000; 4436), phospho-HDAC4 Ser632 (1:1000; 3424), total-HDAC4 (1:1000; 7628), H3 (1:1000; 9715), phospho-eNOS Ser1177 (1:1000; 9517), phospho-eNOS Thr495 (1:1000; 9574), total-eNOS (1: 1000; 32027), and phospho-Histone H2A.X Ser139 (1:1000; 80312) were purchased from Cell Signaling Technology; antibodies against VCAM-1 (1:1000; MA5-31965), IL6 (1:1000; M-620), NADPH oxidase 2 (NOX2) (1:1000; PA5-76034), and VE Cadherin (1:100; PA5-19612) were obtained from Invitrogen; antibody against CD31 (1:100; 14-0311-82) was purchased from eBioscience; antibody against Histone H3ac (Pan-Acetyl) (1:500; sc-518011) was obtained from Santa Cruz Biotechnology; antibody against p16 INK4A (1:100; ZRB1437) was purchased from Merck Millipore; antibody against phospho-HDAC4 Ser632 (1:100; ab39408-1001) and conjugated secondary antibodies against Alexa Fluor ® 488 (1:2000; ab150157) and Alexa Fluor ® 647 (1:2000; ab150075) were obtained from abcam; antibodies against goat anti-rabbit IgG-HRP (1:2000; 4030-05) and goat anti-mouse IgG-HRP (1:2000; 1036-05) were purchased from Southern Biotechnology.

Techniques: Staining, Western Blot, Expressing, Immunofluorescence, Over Expression, Fluorescence, Incubation, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: Gut microbiota-dependent increase in phenylacetic acid induces endothelial cell senescence during aging

doi: 10.1101/2023.11.17.567594

Figure Lengend Snippet: a, Schematic diagram of the experimental setting: PECs were transduced with lentiviral vectors encoding D-aminoacid oxidase (DAAO) targeted to the cell mitochondria for generation of mitochondrial H 2 O 2 in the presence D-alanine (10 mM) for 72 h. Representative images of SA-β-gal staining demonstrates that DAAO-mediated mitochondrial H 2 O 2 induced cellular senescence in PECs, as shown by increased SA-β-gal-positive cells (%) (n=6). b,c, Immunoblots ( b ) and γ-H2A.X immunostaining images ( c ) reveal a marked increase in DDR following mitochondrial H 2 O 2 production in DAAO-transduced PECs incubated with D-alanine (n=6). Scale bar, 20 and 100 μm. Red circles indicate SA-β-gal-positive cells. Data represent triplicated biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: Antibodies against phospho-CaMKII Thr286 (1:1000; 12716), total-CaMKII (1: 1000; 4436), phospho-HDAC4 Ser632 (1:1000; 3424), total-HDAC4 (1:1000; 7628), H3 (1:1000; 9715), phospho-eNOS Ser1177 (1:1000; 9517), phospho-eNOS Thr495 (1:1000; 9574), total-eNOS (1: 1000; 32027), and phospho-Histone H2A.X Ser139 (1:1000; 80312) were purchased from Cell Signaling Technology; antibodies against VCAM-1 (1:1000; MA5-31965), IL6 (1:1000; M-620), NADPH oxidase 2 (NOX2) (1:1000; PA5-76034), and VE Cadherin (1:100; PA5-19612) were obtained from Invitrogen; antibody against CD31 (1:100; 14-0311-82) was purchased from eBioscience; antibody against Histone H3ac (Pan-Acetyl) (1:500; sc-518011) was obtained from Santa Cruz Biotechnology; antibody against p16 INK4A (1:100; ZRB1437) was purchased from Merck Millipore; antibody against phospho-HDAC4 Ser632 (1:100; ab39408-1001) and conjugated secondary antibodies against Alexa Fluor ® 488 (1:2000; ab150157) and Alexa Fluor ® 647 (1:2000; ab150075) were obtained from abcam; antibodies against goat anti-rabbit IgG-HRP (1:2000; 4030-05) and goat anti-mouse IgG-HRP (1:2000; 1036-05) were purchased from Southern Biotechnology.

Techniques: Transduction, Staining, Western Blot, Immunostaining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Melatonin Exerts Prominent, Differential Epidermal and Dermal Anti-Aging Properties in Aged Human Eyelid Skin Ex Vivo

doi: 10.3390/ijms242115963

Figure Lengend Snippet: Overview of the impact of melatonin administration on key skin aging biomarkers. ↑ = increased, ↔ = no change, ↓ = decreased, compared to the vehicle. Mann–Whitney or Student’s t -test, * p < 0.5, ** p < 0.01.

Article Snippet: , γH2A.x , 4% PFA, 10 min at RT , 10% goat serum in PBS , rabbit monoclonal anti-phospho-histone H2A.X (Ser139) (γH2A.X) (1:1500; Cell Signaling, #2577S) , goat monoclonal anti-rabbit IgG-Alexa Fluor 555 (1:400; Invitrogen, A21428).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Melatonin Exerts Prominent, Differential Epidermal and Dermal Anti-Aging Properties in Aged Human Eyelid Skin Ex Vivo

doi: 10.3390/ijms242115963

Figure Lengend Snippet: List of antibodies and immunohistochemical staining treatments used in the study.

Article Snippet: , γH2A.x , 4% PFA, 10 min at RT , 10% goat serum in PBS , rabbit monoclonal anti-phospho-histone H2A.X (Ser139) (γH2A.X) (1:1500; Cell Signaling, #2577S) , goat monoclonal anti-rabbit IgG-Alexa Fluor 555 (1:400; Invitrogen, A21428).

Techniques: Immunohistochemistry, Staining, Blocking Assay, Amplification, Avidin-Biotin Assay