Fig 1: ß-arrestin2 is a target of a2A-AR and knockdown of ß-arrestin2 ameliorates NE-induced tubular cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative images of immunohistochemistry for ß-arrestin2 in sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice. Scale bar, 20 µm. (C, D) Representative Western blotting of ß-arrestin2 (ß-arr2) in sham-operated, UUO/UIRI, and DNx+UUO/UIRI kidneys with densitometry analysis (n=3–6 in each group). (E–G) TKPTS cells were cultured with PBS or NE (10 nM) for 48 hours. (E) Representative images of immunofluorescence for ß-arrestin2 in each group. Scale bar, 20µm. (F) Cells were double-labeled with a2A-AR (green) and ß-arrestin2 (red), and cell nuclei were enhanced by staining with DAPI (blue). Colocalization of a2A-AR (green), and ß-arrestin2 (red) was visualized as yellow in merged images. Scale bar, 20µm. (G) Interactions between ß-arrestin2 and a2A-AR were examined by immunoprecipitation (IP) with an anti-ß-arrestin2 antibody, followed by immunoblotting (IB) with anti-a2A-AR antibodies. (H, I) After an hour of pretreatment with PBS or BRL4408, TKPTS cells were stimulated with NE (10 nM) for 48 hours. (H) Representative Western blotting of ß-arrestin2 (ß-arr2) in TKPTS cells treated with PBS, NE, and NE+BRL (100 nM, 1 µM, 10 µM, and 100 µM) (n=3 in each group). (I) mRNA expression of ß-arrestin2 in PBS, NE, and NE+BRL (10 µM)-treated TKPTS cells as measured by qRT-PCR analysis (n=3–6 in each group). (J, K) TKPTS cells were transfected with scrambled siRNA or three ß-arrestin2 siRNAs (si-Arrb2-1, si-Arrb2-2, or si-Arrb2-3) for 48 hours. ß-arrestin2 (ß-arr2) expression was examined by Western blot analysis (J) and qRT-PCR analysis (K) (n=3 in each group). (L, M) TKPTS cells were transfected with scrambled siRNA (si-control) or ß-arrestin2 siRNA (si-Arrb2) 24 hours before NE (10 nM) treatment. Representative Western blotting of p53 and p21 in each group with densitometry analysis (L) (n=4–5 in each group). mRNA expressions of p21, H2AX, TGF-ß1, IL-6, and IL-8 were tested by qRT-PCR analysis (M) (n=3–6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represented means ± SEM.
Fig 2: ARRB1 participates in Aß25-35-induced autophagy in SH-SY5Y cells. (A) Cells were treated with 20 µM Aß25-35 for 0–16 h, and the levels of ARRB1, ARRB2, and two survival markers (phospho-ERK and phospho-Akt) were detected by western blot. (B) After depletion of arrb1, in the presence of Aß25-35, the expression of ARRB1, autophagy markers (LC3B, Atg7, and Beclin-1) and a survival marker (phospho-ERK) were detected by western blot. (C) With arrb1 knockdown, in presence of Aß25-35, for 1 h, the autophagy marker LC3B was detected by fluorescence staining. The red color indicates immunofluorescence staining of LC3B. The blue color is DAPI, delineating the cell nucleus. The right panel contains the merged pictures. (D) With arrb1 knockdown, in the presence of Aß25-35 for 1 h, the lysosome marker LAMP1 was detected by fluorescence staining. The red color indicates LAMP1 expression, and the blue color is DAPI. The right panel reveals the merge of the two images. (E) With arrb1 knockdown, in the presence of Aß25-35 for 1 h, LC3B was detected by western blot in PC12 cells. (F) With arrb2 knockdown, in the presence of Aß25-35 for 1 h, LC3B was detected by western blot. The raw data of figure A/B/E/F is the figure 3A/3B/3E/3F in supplemental data.
Fig 3: Depletion of ß-arrestin2 aggravates apoptosis of astrocytes stimulated by IL-6. A Cell viability was detected treated with different concentration of IL-6 (0, 100, 200, 300, 400, 500 ng/mL) for primary astrocytes. B, D Apoptosis rate of WT and Arrb2-/- astrocytes were detected by flow cytometry (n = 3). C, E PI/Hoechst staining to observe cell apoptosis stimulated by IL-6 (300 ng/ml). Scale bar: 20 µm n = 7. F Protein levels of Bcl-2 and BAX were detected in WT and Arrb2-/- astrocytes. G, H Protein levels of p-JAK/JAK and p-STAT3/STAT3 were detected in WT and Arrb2-/- astrocytes or transfected with Arrb2 siRNA. I After extraction of nucleus protein from astrocytes, the levels of p-STAT3/STAT3 were detected. J Immunofluorescence staining p-STAT3 in WT and Arrb2-/- astrocytes. p-STAT3: Red; Hoechst: Blue; Scale bar: 20 µm. A Bars and error flags represent the means ± SEM of at least three independent experiments; statistically significant by Student t test; *P < 0.05, **P < 0.01, ***P < 0.001 VS WT Control group or Arrb2-/- untreated group. #P < 0.05, ##P < 0.01 VS corresponding IL-6 stimulated group. D, E Data were denoted as mean ± SEM using two-way ANOVA, then combined with Tukey to assess the differences between groups. ***P < 0.001 VS WT Control group; ###P < 0.001
Fig 4: a2A-AR/ß-arrestin2/NF-?B signaling contributes to mitochondrial dysfunction and cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative Western blotting of VDAC1 and COX IV in each group with densitometry analysis (n=6). (C) Representative Western blotting of VDAC1 and COX IV in TKPTS cells treated with PBS or NE (10 nM) for 48 hours with densitometry analysis (n=3–6 in each group). (D–F) TKPTS cells were transfected with scrambled siRNA (si-control) or ß-arrestin2 siRNA (si-Arrb2) for 24 hours and then incubated with NE (10 nM) for 48 hours. Mitochondrial ultrastructure was observed by transmission electron microscope (D). Scale bar, 1 µm. Mitochondrial mass was tested by MitoTracker deep red probe (100nM) staining (E). Scale bar, 50 µm. Mitochondrial membrane potential was examined by TMRE fluorescent dye (200 nM) (F). Scale bar, 20 µm. (G) Representative Western blotting of NF-?B p65 in TKPTS cells incubated with PBS, NE, and NE+BRL (10 µM) with densitometry analysis (n=4–6 in each group). (H) Representative Western blotting of NF-?B p65 in TKPTS cells of PBS, NE, and NE+si-control, NE+si-Arrb2 with densitometry analysis (n=3-4 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Bars represented means ± SEM.
Fig 5: UNC9995 attenuates apoptosis and inflammation of astrocytes and can be canceled by depletion of ß-arrestin2. A Cell viability of astrocytes pretreated with UNC9995 (1, 5, 10, and 20 µM) for 1 h and then stimulated with IL-6 (300 ng/mL) for 24 h in WT and Arrb2-/- astrocytes. B, C Apoptosis rate was detected by flow cytometry which was pretreated with UNC9995 for 1 h and then stimulated with IL-6 (300 ng/mL) for 24 h in WT and Arrb2-/- astrocytes. Levels of cytokines TNF-a (D) and IL-1ß (E) were detected by ELISA. WT and Arrb2-/- mice were made CUMS models. The time in the center area (F, G), bouts of food sniff (H), latency to sniff (I), and latency to feed (J) in CUMS model mice were detected. K Immunofluorescence staining GFAP in the hippocampus of WT and Arrb2-/- mice. L, M Cytokines of IL-1ß, TNF-a, and IFN-ß in the hippocampus and plasma were detected. N Schmeichel model of activation of ß-arrestin2-biased signaling by UNC9995 in the depression mouse model. Data are analyzed using two-way ANOVA, then combined with Tukey to assess the differences between groups. ns, P > 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 VS WT Control group. #P < 0.05, ##P < 0.01, ###P < 0.001 VS WT IL-6 group or WT CUMS group. $P < 0.05, $$P < 0.01, $$$P < 0.001
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