Journal: bioRxiv

Article Title: AQP1 Differentially Orchestrates Endothelial Cell Senescence

doi: 10.1101/2024.03.13.584782

Figure Lengend Snippet: A , Schematic diagram of the experimental setting: PECs were treated with exogenous H 2 O 2 (25 μM) for 24 h or Baco II (10 μM) for 72 h. Another subset of PECs was transfected with siNeg or siAQP1. At day 3, all cells were then subjected to senescence hallmarks profiling. B, Left, Upper panel, Representative images show significant increase in the numbers of SA-β-gal + cells in the Baco II-and siAQP1-exposed groups, at the magnitude seen in H 2 O 2 –treated PECs, compared to siNeg-transfected PECs. Right, quantitative plots are shown for SA-β-gal + cells (%) (n=6). Left, Lower panel, γ-H2A.X immunostaining represent DDR in Baco II-and siAQP1-exposed PECs compared to siNeg-transfected PECs (n=6). C, qPCR demonstrates that Baco II or siAQP1 treatment resulted in increased expression of CDK inhibitors p16 INK4a , p19 INK4d , and p21 WAF1/Cip1 in PECs. D, qPCR shows that SASP components genes IL1α, IL-1β, and IL-6 are significantly upregulated in Baco II-and siAQP1-exposed PECs (n=6). E, VCAM1 immunostaining shows its marked overexpression in Baco II-and siAQP1-exposed PECs compared to siNeg-transfected PECs (n=6). F, Schematic diagram of the experimental setting: SECs were incubated with exogenous H 2 O 2 (25 μM) for 24 h or Baco II (10 μM) for 72 h. Other SECs were transfected with siNeg or siAQP1. At day 3, all cells were then tested for senescence hallmarks. Left, Upper panel, Baco II or siAQP1 significantly decrease the numbers of SA-β-gal + cells compared to that in siNeg-transfected SECs. Right, quantitative plots are shown for SA-β-gal + cells (%) (n=6). Left, Lower panel, γ-H2A.X immunostaining represent a markedly suppressed DDR in Baco II-and siAQP1-exposed SECs as opposed to that in siNeg-transfected SECs (n=6). G, Baco II or siAQP1 restored cell cycle, represented by reduced p16 INK4a , p19 INK4d , and p21 WAF1/Cip1 transcripts in SECs. H, A significant decrease in transcription of SASP components IL1α, IL-1β, and IL-6 was seen in Baco II-or siAQP1-exposed SECs compared to siNeg-transfected SECs (n=6). I, VCAM1 immunostaining represents its marked downregulation in response to Baco II or siAQP1 in SECs as opposed to that in siNeg-transfected SECs (n=6). Data from in vitro cellular experiments represent triplicated biologically independent experiments. Scale bar, 50 and 200 μm. Error bars represent SD ( B-D, F-H ). P values were calculated using one-way ANOVA followed by Tukey’s post hoc test ( B-D, F-H ). (* 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-AMPKα Thr172 (1:1000; 2535), total-AMPKα (1: 1000; 2532), phospho-eNOS Ser1177 (1:1000; 9517), phospho-eNOS Thr495 (1:1000; 9574), total-eNOS (1: 1000; 32027), Histone H3 (1:1000; 9715), Mef2a (1:1000; 9736), and phospho-Histone H2A.X Ser139 (1:100 and 1:1000; 80312) were purchased from Cell Signaling Technology; antibodies against VCAM-1 (1:100, 1:200 and 1:1000; MA5-31965), and VE Cadherin (1:100; PA5-19612) were obtained from Invitrogen; antibody against AQP1 (1:750 and 1:1000; ab168387), phospho-HDAC4 Ser632 (1:250 and 1:1000; ab39408), and total-HDAC4 (1:1000; ab12172) were obtained from abcam; antibody against CD31 (1:50; DIA-310) was purchased from Dianova; antibody against 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; 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: Transfection, Immunostaining, Expressing, Over Expression, Incubation, In Vitro