Journal: Journal of Biological Chemistry

Article Title: Klotho Inhibits Transforming Growth Factor-β1 (TGF-β1) Signaling and Suppresses Renal Fibrosis and Cancer Metastasis in Mice

doi: 10.1074/jbc.m110.174037

Figure Lengend Snippet: FIGURE 9. Secreted Klotho inhibits EMT in A549 cancer cells. A, Klotho inhibits TGF-1-induced phosphorylation of Smad3 in A549 cells. A549 cells were incubated with secreted Klotho protein for 15 min and then stimulated with TGF-1 for 15 min at the indicated doses. Cell lysates were subjected to immunoblot analyses using antibody against phosphorylated Smad3 (pSmad3) or antibody that recognized Smad3 regardless of its phosphoryla- tion state (Smad3). B, Klotho inhibits TGF-1-induced activation of the Smad- responsive reporter. A549 cells were transfected with a luciferase reporter containing Smad response elements (pGTCT2 2-Luc) and a lacZ expression vector for normalization. These cells were incubated with TGF-1 or Klotho at the indicated doses for 6 h and subjected to a standard luciferase assay. The luciferase activity was normalized with that of non-treated cells. Data indicate means S.E. of three independent experiments. p 0.02 by one-way ANOVA. C, Klotho inhibits TGF-1 binding to A549 cells. TGF-1 binding assays were performed in A549 cells in the absence or presence of Klotho protein (0.1 or 0.3 nM). The amount of bound TGF-1 was normalized with that without Klotho. Data indicate means S.E. of four independent experi- ments. p 0.006 by one-way ANOVA. D, Klotho protein suppresses TGF-1- induced decrease in E-cadherin and increase in N-cadherin. A549 cells were treated with TGF-1 and/or Klotho at the indicated doses for 6 h and then subjected to immunoblot analyses 48 h later. E, Klotho protein suppresses TGF-1-induced cell migration. A549 cells were treated with TGF-1 and/or Klotho at the indicated doses for 6 h and then subjected to a standard Tran- swell migration assay. Data indicate means S.E. of three independent experiments. *, p 0.001 versus TGF-1 treatment alone by two-tailed t test.

Article Snippet: Primary antibodies used in this study were Klotho (KM2119)(30), SMA, Vimentin (Santa Cruz Biotechnology), E-Cadherin (BD Biosciences, San Jose, CA), N-cadherin (Santa Cruz Biotechnology), phosphorylated Smad2 (Cell Signaling Technology, Beverley, CA), phosphorylated Smad3 (Cell Signaling), Smad2 (Cell Signaling), Smad3 (Cell Signaling), GAPDH (Abcam).

Techniques: Phospho-proteomics, Incubation, Western Blot, Activation Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Binding Assay, Migration, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Tartrate-Resistant Acid Phosphatase 5/ACP5 Interacts with p53 to Control the Expression of SMAD3 in Lung Adenocarcinoma

doi: 10.1016/j.omto.2020.01.010

Figure Lengend Snippet: ACP5 Regulated EMT in LUAD Cells (A) The morphology of A549 cells treated with TGF-β1 (10 ng/mL) for different time intervals was visualized by phase-contrast microscopy. Original magnification, ×200; scale bars, 100 μm. An immunofluorescence staining assay showed the results of rhodamine-phalloidin staining. Actin microfilaments were stained by rhodamine-conjugated phalloidin. (B) Immunofluorescence staining assays were used to evaluate the expressions of E-cadherin and vimentin upon ACP5 knockdown in A549 cells. Cell nuclei were visualized by DAPI. Original magnification, ×400; scale bars, 50 μm. (C) ACP5 in A549 and H1975 cells were knocked down or overexpressed treated with or without 10 ng/mL TGF-β1 for 24 h. The levels of fibronectin, E-cadherin, and vimentin were determined by western blot. The results are summarized as the mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by independent Student’s t test.

Article Snippet: Subsequently, the cells were incubated with primary antibodies against mouse-originated vimentin antibody (R&D Systems, Minneapolis, MN, USA, 1:100), mouse-originated E-cadherin antibody (R&D Systems, Minneapolis, MN, USA, 1:100), mouse-originated p53 antibody (Cell Signaling Technology, Danvers, MA, USA, 1:50), and rabbit-originated ACP5 antibody (Proteintech, Wuhan, China, 1:100) overnight at 4°C and then incubated with an Alexa Fluor 488-labeled anti-mouse antibody and an Alexa Fluor 594-conjugated anti-rabbit antibody (Abbkine, Redlands, CA, USA, 1:400) for 1 h, as instructed.

Techniques: Microscopy, Immunofluorescence, Staining, Knockdown, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Tartrate-Resistant Acid Phosphatase 5/ACP5 Interacts with p53 to Control the Expression of SMAD3 in Lung Adenocarcinoma

doi: 10.1016/j.omto.2020.01.010

Figure Lengend Snippet: ACP5 Promoted Xenograft Tumor Growth and Enhanced Metastatic Potential in Mouse Models (A–F) Nude mice were subcutaneously injected with A549 cells stably overexpressed for ACP5 (A549-ACP5) or control (A549-Vec) cells. (A) Images of the tumor lumps from the indicated groups at the endpoint of the experiment. n = 10 mice per group. (B) Tumor formation in nude mice treated with A549-ACP5 or A549-Vec cells was monitored at the indicated time points. (C) Tumor weights were measured at the last time point. (D) Quantitative real-time PCR for ACP5 expression in xenograft tumor tissues of A549-Vec (n = 5) and A549-ACP5 groups (n = 5). (E) Representative H&E staining images (top panel) (original magnification, ×100; scale bars, 200 μm) and IHC images of ACP5 staining (bottom panel) (original magnification, ×400; scale bars, 50 μm) in sections of xenograft tumor of A549 cells. (F) The expression of E-cadherin, p53, SMAD3, and ACP5 was detected by western blot in xenograft tumors from the A549-Vec (n = 5) and A549-ACP5 (n = 5) groups. (G–I) A549-ACP5 or A549-Vec cells were intravenously injected into nude mice and lung tissues were isolated after 5 weeks (A549-ACP5, n = 8; A549-vector, n = 10). (G) Representative lung images showing metastases in the lung. (H) Number of the metastatic nodules visible to the naked eye in the lung of mice. (I) H&E staining (original magnification, ×100; scale bars, 200 μm) and IHC staining for ACP5 (original magnification, 400; scale bars, 50 μm) performed with sections of metastatic lung nodules. (J) Schematic illustration of the role of ACP5 in LUAD. The results are expressed as the mean ± SD.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by independent Student’s t test.

Article Snippet: Subsequently, the cells were incubated with primary antibodies against mouse-originated vimentin antibody (R&D Systems, Minneapolis, MN, USA, 1:100), mouse-originated E-cadherin antibody (R&D Systems, Minneapolis, MN, USA, 1:100), mouse-originated p53 antibody (Cell Signaling Technology, Danvers, MA, USA, 1:50), and rabbit-originated ACP5 antibody (Proteintech, Wuhan, China, 1:100) overnight at 4°C and then incubated with an Alexa Fluor 488-labeled anti-mouse antibody and an Alexa Fluor 594-conjugated anti-rabbit antibody (Abbkine, Redlands, CA, USA, 1:400) for 1 h, as instructed.

Techniques: Injection, Stable Transfection, Control, Real-time Polymerase Chain Reaction, Expressing, Staining, Western Blot, Isolation, Plasmid Preparation, Immunohistochemistry