Journal: Autophagy

Article Title: TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

doi: 10.1080/15548627.2016.1204496

Figure Lengend Snippet: Figure 3. Autophagy induction was critical for mediating VEGFA production in Beas-2B cells upon PM2.5 exposure. (A) Beas-2B cells were pretreated with 3-MA, followed by exposure to PM2.5 (100 mg/mL). Then, the expression of VEGFA, BECN1 and MAP1LC3B was analyzed 24 h after PM2.5 exposure. (B) Beas-2B cells stably transfected with VEGFA promoter-driven luciferase reporter were treated with 3-MA and PM2.5 as described in (A). Then, the induction of VEGFA promoter-dependent luciferase activ- ity was examined 12 h after PM2.5 exposure (, P < 0.01). (C) Beas-2B cells were treated with 3-MA and PM2.5 as described in (A). Then, the production and secretion of VEGFA in Beas-2B cells were detected in the cell culture supernatant using ELISA 24 h after PM2.5 exposure (, P < 0.01). (D) Beas-2B cells were transfected with ATG5 siRNA or control siRNA and then exposed to PM2.5 (100 mg/mL) 36 h after transfection. The expression of ATG5, VEGFA, BECN1 and MAP1LC3B was examined 24 h after PM2.5 exposure. (E) Beas-2B cells stably transfected with VEGFA promoter-driven luciferase reporter were transfected with ATG5 siRNA or control siRNA and treated with PM2.5 as described in (D). Then, the induction of VEGFA promoter-dependent luciferase activity was determined 12 h after PM2.5 exposure (, P < 0.01). (F) Beas-2B cells were transfected and treated with PM2.5 as described in (D). Then, the production and secretion of VEGFA in Beas-2B cells were detected in the cell culture supernatant using ELISA 24 h after PM2.5 exposure (, P < 0.01). (G) Beas-2B cells were transfected with BECN1 siRNA or control siRNA and then exposed to PM2.5 (100 mg/mL) 36 h after transfection. The expression of BECN1, MAP1LC3B and VEGFA was examined 24 h after PM2.5 exposure. (H) Beas-2B cells stably transfected with VEGFA promoter- driven luciferase reporter were transfected with BECN1 siRNA or control siRNA and treated with PM2.5 as described in (G). Then, the induction of VEGFA promoter-depen- dent luciferase activity was determined 12 h after PM2.5 exposure (, P < 0.01). (I) Beas-2B cells were transfected and treated with PM2.5 as described in (G). Then, the production and secretion of VEGFA in Beas-2B cells were detected in the cell culture supernatant using ELISA 24 h after PM2.5 exposure (, P < 0.01). (J) Beas-2B cells were treated as described in Fig. 2F, and then VEGFA expression levels were measured 24 h after PM2.5 exposure. (K) Beas-2B cells stably transfected with VEGFA pro- moter-driven luciferase reporter were treated as in Fig. 2F, and the induction of VEGFA promoter-dependent luciferase activity was examined 12 h after PM2.5 exposure (, P < 0.01). (L) Beas-2B cells were treated as described in Fig. 2F, and then the production and secretion of VEGFA in Beas-2B cells were detected in the cell culture supernatant using ELISA 24 h after PM2.5 exposure (, P < 0.01).

Article Snippet: The siRNAs and regents used were as follows: ATR siRNA (Cell Signaling Technology, 6288), ATM siRNA (Cell Signaling Technology, 6328), ATG5 siRNA (Cell Signaling Technology, 6348), DRAM1 siRNA (Riobo Technology, 1314.14), CHEK1 (CHK1) siRNA (Riobo Technology, 13285.14), BECN1 siRNA (Cell Signaling Technology, 6222), 3- MA (Sigma-Aldrich, M9281), PMB (Sigma-Aldrich, P1004) and BafA1 (LC Laboratories, B1080); Primary antibodies used were as follows: BECN1 (Cell Signaling Technology, 3495), MAP1LC3B (Cell Signaling Technology, 3868), phospho-TP53 (Ser15; Cell Signaling Technology, 9284), TP53 (Cell Signaling Technology, 2524), phospho-SRC (Tyr416; Cell Signaling Technology, 6943), SRC (Cell Signaling Technology, 2109), phospho-STAT3 (Tyr705; Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), phospho-ATM (Ser1981; Cell Signaling Technology, 5883), ATM (Cell Signaling Technology, 2873), phospho-ATR (Ser428; Cell Signaling Technology, 2853), ATR (Cell Signaling Technology, 2790), phospho-CHEK1 (Ser345; Cell Signaling Technology, 2348), CHEK1 (Cell Signaling Technology, 2360), SQSTM1 (Cell Signaling Technology, 8025), ACTB (Cell Signaling Technology, 4970), ATG5 (Cell Signaling Technology, 9980) and DRAM1 (Santa Cruz Biotechnology, 98654).

Techniques: Expressing, Stable Transfection, Transfection, Luciferase, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Activity Assay

Journal: Autophagy

Article Title: TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

doi: 10.1080/15548627.2016.1204496

Figure Lengend Snippet: Figure 4. PM2.5-induced autophagy contributed to VEGFA production by activating the SRC-STAT3 pathway. (A) Beas-2B cells were left untreated or were treated with PM2.5 as described in Fig. 1A; then, the activation status of SRC and STAT3 was determined. (B) Beas-2B cells were transfected with STAT3-dependent luciferase reporter, and stable transfectants were established. The transfectants were exposed to different doses of PM2.5 (as indicated), and the induction of STAT3-dependent luciferase activity was examined 12 h after PM2.5 exposure (, P < 0.05; , P < 0.01). (C) Beas-2B cells were transfected with STAT3 siRNA or control siRNA; then, cells were treated with PM2.5 (100 mg/mL) 36 h after transfection. The expression of STAT3 and VEGFA was examined 24 h after PM2.5 exposure. (D) Beas-2B cells were transfected with SRC siRNA or control siRNA and then were treated with PM2.5 (100 mg/mL) 36 h after transfection. The expressions of SRC and VEGFA and the activation status of STAT3 was examined 24 h after PM2.5 exposure. (E) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with SRC siRNA or control siRNA and exposed to PM2.5 (100 mg/mL) 36 h after transfection. The induction of the STAT3-dependent luciferase activity was determined 12 h after PM2.5 exposure (, P < 0.01). (F) Beas-2B cells were treated as described in Fig. 1F, and then the activation status of SRC and STAT3 was determined 24 h after PM2.5 exposure. (H, I and J) Beas-2B cells were treated or transfected as described in Fig. 3A, 3C and 3E. Then, the activation status of SRC and STAT3 was determined 24 h after PM2.5 exposure. p, phosphorylated.

Article Snippet: The siRNAs and regents used were as follows: ATR siRNA (Cell Signaling Technology, 6288), ATM siRNA (Cell Signaling Technology, 6328), ATG5 siRNA (Cell Signaling Technology, 6348), DRAM1 siRNA (Riobo Technology, 1314.14), CHEK1 (CHK1) siRNA (Riobo Technology, 13285.14), BECN1 siRNA (Cell Signaling Technology, 6222), 3- MA (Sigma-Aldrich, M9281), PMB (Sigma-Aldrich, P1004) and BafA1 (LC Laboratories, B1080); Primary antibodies used were as follows: BECN1 (Cell Signaling Technology, 3495), MAP1LC3B (Cell Signaling Technology, 3868), phospho-TP53 (Ser15; Cell Signaling Technology, 9284), TP53 (Cell Signaling Technology, 2524), phospho-SRC (Tyr416; Cell Signaling Technology, 6943), SRC (Cell Signaling Technology, 2109), phospho-STAT3 (Tyr705; Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), phospho-ATM (Ser1981; Cell Signaling Technology, 5883), ATM (Cell Signaling Technology, 2873), phospho-ATR (Ser428; Cell Signaling Technology, 2853), ATR (Cell Signaling Technology, 2790), phospho-CHEK1 (Ser345; Cell Signaling Technology, 2348), CHEK1 (Cell Signaling Technology, 2360), SQSTM1 (Cell Signaling Technology, 8025), ACTB (Cell Signaling Technology, 4970), ATG5 (Cell Signaling Technology, 9980) and DRAM1 (Santa Cruz Biotechnology, 98654).

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Control, Expressing, Stable Transfection

Journal: Autophagy

Article Title: TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

doi: 10.1080/15548627.2016.1204496

Figure Lengend Snippet: Figure 5. PM2.5 induced TP53 transactivation, which was critical for mediating autophagy induction in Beas-2B cells. (A and B) Beas-2B cells were left untreated or were treated with PM2.5 as described in Fig. 1A and 1E, and the induction of TP53 activation and DRAM1 expression was examined. (C) Beas-2B cells were transfected with TP53-dependent luciferase reporter, and stable transfectants were established. The transfectants were exposed to different doses of PM2.5 (as indicated), and the induc- tion of TP53-dependent luciferase activity was examined 12 h after PM2.5 exposure (, P < 0.01). (D) Beas-2B cells were treated as described in Fig. 1F, and the induction of TP53 activation and DRAM1 expression was examined 24 h after PM2.5 exposure. (E) Beas-2B cells were transfected with TP53 siRNA, DRAM1 siRNA or control siRNA; then, they were treated with PM2.5 (100 mg/mL) 36 h after transfection. The activation status of TP53 and the expression levels of DRAM1, BECN1 MAP1LC3B were exam- ined 24 h after PM2.5 exposure. (F and G) Beas-2B cells were transfected and treated as described in (E), and then the autophagy signals were detected as described in Fig. 2C and 2D (, P < 0.01). p, phosphorylated.

Article Snippet: The siRNAs and regents used were as follows: ATR siRNA (Cell Signaling Technology, 6288), ATM siRNA (Cell Signaling Technology, 6328), ATG5 siRNA (Cell Signaling Technology, 6348), DRAM1 siRNA (Riobo Technology, 1314.14), CHEK1 (CHK1) siRNA (Riobo Technology, 13285.14), BECN1 siRNA (Cell Signaling Technology, 6222), 3- MA (Sigma-Aldrich, M9281), PMB (Sigma-Aldrich, P1004) and BafA1 (LC Laboratories, B1080); Primary antibodies used were as follows: BECN1 (Cell Signaling Technology, 3495), MAP1LC3B (Cell Signaling Technology, 3868), phospho-TP53 (Ser15; Cell Signaling Technology, 9284), TP53 (Cell Signaling Technology, 2524), phospho-SRC (Tyr416; Cell Signaling Technology, 6943), SRC (Cell Signaling Technology, 2109), phospho-STAT3 (Tyr705; Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), phospho-ATM (Ser1981; Cell Signaling Technology, 5883), ATM (Cell Signaling Technology, 2873), phospho-ATR (Ser428; Cell Signaling Technology, 2853), ATR (Cell Signaling Technology, 2790), phospho-CHEK1 (Ser345; Cell Signaling Technology, 2348), CHEK1 (Cell Signaling Technology, 2360), SQSTM1 (Cell Signaling Technology, 8025), ACTB (Cell Signaling Technology, 4970), ATG5 (Cell Signaling Technology, 9980) and DRAM1 (Santa Cruz Biotechnology, 98654).

Techniques: Activation Assay, Expressing, Transfection, Luciferase, Activity Assay, Control

Journal: Autophagy

Article Title: TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

doi: 10.1080/15548627.2016.1204496

Figure Lengend Snippet: Figure 6. ATR was required for the induction of TP53-dependent autophagy in Beas-2B cells upon PM2.5 exposure. (A) Beas-2B cells were treated as described in Fig. 1A, and then the activation status of ATM and ATR was determined. (B) Beas-2B cells were transfected with ATR siRNA, ATM siRNA or control siRNA; then, cells were treated with PM2.5 (100 mg/mL) 36 h after transfection. The activation status of TP53 and the expression levels of ATR, ATM, DRAM1, BECN1 and MAP1LC3B were examined 24 h after PM2.5 exposure. (C) Beas-2B cells stably transfected with TP53-dependent luciferase reporter were transfected with ATR siRNA, ATM siRNA or control siRNA; then exposed to PM2.5 (100 mg/mL) 36 h after transfection. The induction of TP53-dependent luciferase activity was determined 12 h after PM2.5 exposure (, P < 0.01). (D and E) Beas-2B cells were transfected with ATR siRNA or control siRNA and then treated with PM2.5 (100 mg/mL) 36 h after transfection. The autophagy signals were detected as described in Fig. 2C and 2D (, P < 0.01). p, phosphorylated.

Article Snippet: The siRNAs and regents used were as follows: ATR siRNA (Cell Signaling Technology, 6288), ATM siRNA (Cell Signaling Technology, 6328), ATG5 siRNA (Cell Signaling Technology, 6348), DRAM1 siRNA (Riobo Technology, 1314.14), CHEK1 (CHK1) siRNA (Riobo Technology, 13285.14), BECN1 siRNA (Cell Signaling Technology, 6222), 3- MA (Sigma-Aldrich, M9281), PMB (Sigma-Aldrich, P1004) and BafA1 (LC Laboratories, B1080); Primary antibodies used were as follows: BECN1 (Cell Signaling Technology, 3495), MAP1LC3B (Cell Signaling Technology, 3868), phospho-TP53 (Ser15; Cell Signaling Technology, 9284), TP53 (Cell Signaling Technology, 2524), phospho-SRC (Tyr416; Cell Signaling Technology, 6943), SRC (Cell Signaling Technology, 2109), phospho-STAT3 (Tyr705; Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), phospho-ATM (Ser1981; Cell Signaling Technology, 5883), ATM (Cell Signaling Technology, 2873), phospho-ATR (Ser428; Cell Signaling Technology, 2853), ATR (Cell Signaling Technology, 2790), phospho-CHEK1 (Ser345; Cell Signaling Technology, 2348), CHEK1 (Cell Signaling Technology, 2360), SQSTM1 (Cell Signaling Technology, 8025), ACTB (Cell Signaling Technology, 4970), ATG5 (Cell Signaling Technology, 9980) and DRAM1 (Santa Cruz Biotechnology, 98654).

Techniques: Activation Assay, Transfection, Control, Expressing, Stable Transfection, Luciferase, Activity Assay

Journal: Autophagy

Article Title: TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

doi: 10.1080/15548627.2016.1204496

Figure Lengend Snippet: Figure 7. CHEK1 was the downstream target of ATR that mediated TP53-dependent autophagy in Beas-2B cells under PM2.5 exposure. (A) Beas-2B cells were treated as described in Fig. 1A, and the activation status of CHEK1 was determined. (B) Beas-2B cells were transfected with ATR siRNA or control siRNA and treated with PM2.5 (100 mg/mL) 36 h after transfection. The activation status of CHEK1 was determined 24 h after PM2.5 exposure. (C) Beas-2B cells were treated as described in Fig. 1F, and the activation status of the ATR-CHEK1 axis was determined 24 h after PM2.5 exposure. (D) Beas-2B cells were transfected with CHEK1 siRNA or control siRNA and then treated with PM2.5 (100 mg/mL) 36 h after transfection. The activation status of CHEK1 and TP53 and the expression levels of DRAM1, BECN1 and MAP1LC3B were exam- ined 24 h after PM2.5 exposure. (E) Beas-2B cells stably transfected with TP53-dependent luciferase reporter were transfected with CHEK1 siRNA or control siRNA and exposed to PM2.5 (100 mg/mL) 36 h after transfection. The induction of TP53-dependent luciferase activity was determined 12 h after PM2.5 exposure (, P < 0.01). (F and G) Beas-2B cells were transfected with CHEK1 siRNA or control siRNA and treated with PM2.5 (100 mg/mL) 36 h after transfection. Then, the autophagy signals were detected as described in Fig. 2C and 2D (, P < 0.01). p, phosphorylated.

Article Snippet: The siRNAs and regents used were as follows: ATR siRNA (Cell Signaling Technology, 6288), ATM siRNA (Cell Signaling Technology, 6328), ATG5 siRNA (Cell Signaling Technology, 6348), DRAM1 siRNA (Riobo Technology, 1314.14), CHEK1 (CHK1) siRNA (Riobo Technology, 13285.14), BECN1 siRNA (Cell Signaling Technology, 6222), 3- MA (Sigma-Aldrich, M9281), PMB (Sigma-Aldrich, P1004) and BafA1 (LC Laboratories, B1080); Primary antibodies used were as follows: BECN1 (Cell Signaling Technology, 3495), MAP1LC3B (Cell Signaling Technology, 3868), phospho-TP53 (Ser15; Cell Signaling Technology, 9284), TP53 (Cell Signaling Technology, 2524), phospho-SRC (Tyr416; Cell Signaling Technology, 6943), SRC (Cell Signaling Technology, 2109), phospho-STAT3 (Tyr705; Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), phospho-ATM (Ser1981; Cell Signaling Technology, 5883), ATM (Cell Signaling Technology, 2873), phospho-ATR (Ser428; Cell Signaling Technology, 2853), ATR (Cell Signaling Technology, 2790), phospho-CHEK1 (Ser345; Cell Signaling Technology, 2348), CHEK1 (Cell Signaling Technology, 2360), SQSTM1 (Cell Signaling Technology, 8025), ACTB (Cell Signaling Technology, 4970), ATG5 (Cell Signaling Technology, 9980) and DRAM1 (Santa Cruz Biotechnology, 98654).

Techniques: Activation Assay, Transfection, Control, Expressing, Stable Transfection, Luciferase, Activity Assay

Journal: Autophagy

Article Title: TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

doi: 10.1080/15548627.2016.1204496

Figure Lengend Snippet: Figure 8. ATR-CHEK1-TP53 signaling pathway activation was required for mediating autophagy-dependent VEGFA production in Beas-2B cells under PM2.5 exposure. (A, C and E) Beas-2B cells were transfected with TP53 siRNA or DRAM1 siRNA (A), ATR siRNA (C) or CHEK1 siRNA (E) and their respective control siRNAs; then, cells were treated with PM2.5 (100 mg/mL) 36 h after transfection. The activation status of SRC and STAT3 and the expression level of VEGFA were determined 24 h after PM2.5 exposure. (B, D and F) Beas-2B cells stably transfected with a VEGFA promoter-driven luciferase reporter were transfected and treated as described in (A, C and E), respec- tively. Then, the induction of VEGFA promoter-dependent luciferase activity was determined 12 h after PM2.5 exposure (, P < 0.01). p, phosphorylated.

Article Snippet: The siRNAs and regents used were as follows: ATR siRNA (Cell Signaling Technology, 6288), ATM siRNA (Cell Signaling Technology, 6328), ATG5 siRNA (Cell Signaling Technology, 6348), DRAM1 siRNA (Riobo Technology, 1314.14), CHEK1 (CHK1) siRNA (Riobo Technology, 13285.14), BECN1 siRNA (Cell Signaling Technology, 6222), 3- MA (Sigma-Aldrich, M9281), PMB (Sigma-Aldrich, P1004) and BafA1 (LC Laboratories, B1080); Primary antibodies used were as follows: BECN1 (Cell Signaling Technology, 3495), MAP1LC3B (Cell Signaling Technology, 3868), phospho-TP53 (Ser15; Cell Signaling Technology, 9284), TP53 (Cell Signaling Technology, 2524), phospho-SRC (Tyr416; Cell Signaling Technology, 6943), SRC (Cell Signaling Technology, 2109), phospho-STAT3 (Tyr705; Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), phospho-ATM (Ser1981; Cell Signaling Technology, 5883), ATM (Cell Signaling Technology, 2873), phospho-ATR (Ser428; Cell Signaling Technology, 2853), ATR (Cell Signaling Technology, 2790), phospho-CHEK1 (Ser345; Cell Signaling Technology, 2348), CHEK1 (Cell Signaling Technology, 2360), SQSTM1 (Cell Signaling Technology, 8025), ACTB (Cell Signaling Technology, 4970), ATG5 (Cell Signaling Technology, 9980) and DRAM1 (Santa Cruz Biotechnology, 98654).

Techniques: Activation Assay, Transfection, Control, Expressing, Stable Transfection, Luciferase, Activity Assay

Journal: The Journal of general virology

Article Title: HBx natural variants containing Ser-101 instead of Pro-101 evade ubiquitin-dependent proteasomal degradation by activating proteasomal activator 28 gamma expression.

doi: 10.1099/jgv.0.001337

Figure Lengend Snippet: Fig. 2. HBx variants containing Ser-101 stabilize p53 by activating the ATM–CHK2 pathway. HepG2 cells were transiently transfected with an empty vector or HBx expression plasmid for 48 h. (a) The levels of intracellular ROS were determined by staining with the redox-sensitive dye CM-H2DCFDNA, as described previously [47, 48]. The ROS levels are presented as the relative fluorescent signal per microgram of protein. The data represent the mean±sd from five independent experiments (n=5). (b) Cells were either mock-treated or treated with 10 µM KU-55933 for 1 h before harvesting, followed by Western blotting. (c) The HA-Ub expression plasmid was included in the transfection reactions. Cells were treated with 10 µM MG132 for 4 h before harvesting. Total p53 proteins were immunoprecipitated with an anti-p53 antibody and subjected to Western blotting using anti-p53, anti-HA and anti-MDM antibodies to detect p53, HA-Ub- complexed p53 and MDM2, respectively. The input shows levels of the indicated proteins in whole-cell lysates. (d) HepG2 cells were transfected with either an empty vector or the indicated HBx expression plasmid for 44 h and then treated with 10 µM MG132 for an additional 4 h, followed by Western blotting. (e) HepG2 cells were transfected with either an empty vector or the indicated HBx expression plasmid, followed by Western blotting to measure the levels of p53, p21, PUMA, Bax, HBx and γ-tubulin.

Article Snippet: Membranes were then incubated with primary antibodies against p21, p53, ATM, Bax, β-Gal, CHK2, HBc, HBs, MDM2, NTCP, PA28γ, PUMA, Siah-1 and Ub (Santa Cruz Biotechnology); pSer1981 ATM, pSer-15 p53, pSer-20 p53 and pThr-68 CHK2 (Cell Signaling); HBx (Millipore); HA (Roche) to detect HA-tagged HBx; and γ-tubulin (Sigma), and subsequently with an appropriate horseradish peroxidase-conjugated secondary antibody: anti-mouse, anti-goat, or anti-rabbit IgG (H+L)-HRP (Bio-Rad).

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Western Blot, Immunoprecipitation