Journal: Autophagy

Article Title: NUPR1 maintains autolysosomal efflux by activating SNAP25 transcription in cancer cells

doi: 10.1080/15548627.2017.1338556

Figure Lengend Snippet: NUPR1 depletion causes premature senescence in vitro and represses tumorigenesis in vivo. (A) Representative images of GLB1 activity in NUPR1-depleted and control cells as indicated (left panels). Quantification of GLB1-positive cells was determined in 10 different fields from 3 independent experiments (mean ± SEM) (right panel). Scale bars: 10 µm. (B) NUPR1-depleted A549 cells were collected for cell cycle analysis by flow cytometry. The percentage of cells in G0/G1, S, and G2/M phases from 3 independent experiments is shown (right panel, mean ± SEM). (C) Western blot analysis of the indicated proteins in A549 and H460 cells infected with NUPR1 shRNA, with ACTB as a loading control. (D) Cellular proliferation of control and NUPR1-shRNA A549 cells was assessed using a 5-bromodeoxyuridine (BrdU) assay. The data are represented as the mean ± SEM of 6 experiments. (E) Clonogenic assays performed with control and NUPR1-shRNA A549 cells. A total of 1,500 cells were seeded in 24-well plates and grown for 2 wk. The graph shows the quantification of the mean number of colonies at different time point as indicated. ** P < 0.01 compared to control. (F) Western blot analysis of CASP3, cleaved CASP3, CASP7, CASP9, and ACTB in NUPR1-depleted A549 cells. (G) Western blot analysis of CDKN1B in A549 cells by NUPR1 depletion and/or its reexpression, with ACTB as a loading control. (H) A549 cells with lentivirus-delivered NUPR1 knockdown were subcutaneously implanted into female athymic nude mice (n = 6 for each experimental condition). The tumor image (left panel) on d 24 and tumor growth curve (right panel, mean ± SEM) are shown. ** P < 0.01 compared to control.

Article Snippet: DNA sequences for shRNA are listed in Table S2 . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary Antibody Dilution Ratio Supplier ATG5 (rabbit polyclonal) 1:1,000 Cell Signaling Technology, 2630 ACTB (mouse monoclonal) 1:3,000 Sigma-Aldrich, A-3853 BECN1(rabbit polyclonal) 1:2,000 Cell Signaling Technology, 3738 CASP3 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 9665 CDKN1A/p21Waf1 (rabbit monoclonal) 1:2,000 Cell Signaling Technology, 9932 CDKN1B/p27Kip1(rabbit monoclonal) 1:2,000 Cell Signaling Technology, 9932 CDKN2A/p16INK4a (rabbit monoclonal) 1:2,000 Abcam, ab108349 CTSB (mouse monoclonal) 1:1,000 Santa Cruz Biotechnology, sc-365558 CTSD (mouse monoclonal) 1:1,000 Santa Cruz Biotechnology, sc-377299 Cleaved CASP3 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 9664 Cleaved CASP7 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 8438 Cleaved CASP9 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 7237 FLAG (mouse monoclonal) 1:4,000 Sigma-Aldrich, F3165 GAPDH (rabbit polyclonal) 1:3,000 Trevigen, 2275 HA (rabbit monoclonal) 1:2.000 Cell Signaling Technology, 3724S LC3B (rabbit polyclonal) 1:2,000 Sigma-Aldrich, L7543 MTOR (rabbit monoclonal) 1:2,000 Cell Signaling Technology, 2983 NUPR1 (mouse monoclonal) 1:1,000 Abcam, ab87454 p-MTOR(S2448) (rabbit monoclonal) 1:2,000 Cell Signaling Technology, 5536 PCNA (rabbit polyclonal) 1:1,000 Abcam, ab18197 SNAP25 (rabbit polyclonal) 1:1,000 Abcam, ab109105 SQSTM1/p62 (rabbit polyclonal) 1:1,000 Cell Signaling Technology, 7695 VAMP8 (mouse monoclonal) 1:1,000 Santa Cruz Biotechnology, sc-166820 Open in a separate window Primary antibodies used in this study.

Techniques: In Vitro, In Vivo, Activity Assay, Cell Cycle Assay, Flow Cytometry, Western Blot, Infection, shRNA, BrdU Staining

Journal: Autophagy

Article Title: NUPR1 maintains autolysosomal efflux by activating SNAP25 transcription in cancer cells

doi: 10.1080/15548627.2017.1338556

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: DNA sequences for shRNA are listed in Table S2 . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary Antibody Dilution Ratio Supplier ATG5 (rabbit polyclonal) 1:1,000 Cell Signaling Technology, 2630 ACTB (mouse monoclonal) 1:3,000 Sigma-Aldrich, A-3853 BECN1(rabbit polyclonal) 1:2,000 Cell Signaling Technology, 3738 CASP3 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 9665 CDKN1A/p21Waf1 (rabbit monoclonal) 1:2,000 Cell Signaling Technology, 9932 CDKN1B/p27Kip1(rabbit monoclonal) 1:2,000 Cell Signaling Technology, 9932 CDKN2A/p16INK4a (rabbit monoclonal) 1:2,000 Abcam, ab108349 CTSB (mouse monoclonal) 1:1,000 Santa Cruz Biotechnology, sc-365558 CTSD (mouse monoclonal) 1:1,000 Santa Cruz Biotechnology, sc-377299 Cleaved CASP3 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 9664 Cleaved CASP7 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 8438 Cleaved CASP9 (rabbit polyclonal) 1:1000 Cell Signaling Technology, 7237 FLAG (mouse monoclonal) 1:4,000 Sigma-Aldrich, F3165 GAPDH (rabbit polyclonal) 1:3,000 Trevigen, 2275 HA (rabbit monoclonal) 1:2.000 Cell Signaling Technology, 3724S LC3B (rabbit polyclonal) 1:2,000 Sigma-Aldrich, L7543 MTOR (rabbit monoclonal) 1:2,000 Cell Signaling Technology, 2983 NUPR1 (mouse monoclonal) 1:1,000 Abcam, ab87454 p-MTOR(S2448) (rabbit monoclonal) 1:2,000 Cell Signaling Technology, 5536 PCNA (rabbit polyclonal) 1:1,000 Abcam, ab18197 SNAP25 (rabbit polyclonal) 1:1,000 Abcam, ab109105 SQSTM1/p62 (rabbit polyclonal) 1:1,000 Cell Signaling Technology, 7695 VAMP8 (mouse monoclonal) 1:1,000 Santa Cruz Biotechnology, sc-166820 Open in a separate window Primary antibodies used in this study.

Techniques: