Journal: Molecular and cellular biochemistry

Article Title: Early-life bisphenol A exposure causes neuronal pyroptosis in juvenile and adult male rats through the NF-κB/IL-1β/NLRP3/caspase-1 signaling pathway: exploration of age and dose as effective covariates using an in vivo and in silico modeling approach.

doi: 10.1007/s11010-024-05039-4

Figure Lengend Snippet: Fig. 8 The molecular docking analysis of BPA and neuro- inflammatory, autophagic as well as, pyroptotic molecules displaying 2D and 3D binding interactions of BPA [PubChem CID: 6623] against A NF-kB [PDB ID: 1NFK], B IL-1β [PDB ID: 2MIB], C IL-2 [PDB ID: 4YQX], D IL-12 [PDB ID: 3HMX], E COX-2 [PDB ID: 1PXX], F NLRP3 [PDB ID: 7vtq], G Beclin-1 [PDB ID: 2PON], H LC3A [PDB ID: 6TBE], I LC3B [PDB ID: 5XAC], J Caspase-1 [PDB ID: 6VIE]. The green dotted lines denote hydrogen bonds between ligand and aminoacids, whereas bink/purple dotted lines repre- sent hydrophobic interactions. Electrostatic interactions are shown as orange dotted lines. The red dotted line indicate an unfavorable donor-donor

Article Snippet: Fine Test (cat no. ER1965) provided the NLRP3 kit, LSBio (cat no. LS-F9917; LS-F19802) provided the LC3A and LC3B kits, respectively, and Cusabio (cat no. CSB-EL002658RA provided the beclin-1 kit.

Techniques: Binding Assay

Journal: Antioxidants

Article Title: New Amides and Phosphoramidates Containing Selenium: Studies on Their Cytotoxicity and Antioxidant Activities in Breast Cancer

doi: 10.3390/antiox10040590

Figure Lengend Snippet: Western blot analysis of autophagy markers SQSTM1/p62, Beclin-1, p-JNKm p-AMPK, LC3B and p53 of MCF-7 cell culture treated with I.1f. ** statistical significance with values of p < 0.01; *** statistical significance with values of p < 0.001.

Article Snippet: Primary specific antibodies were incubated in 5% milk-TBS-Tween-20 (1 h, room temperature) to detect LC3B, Beclin-1 (D40C5), SQSTM1/p62, AMPK, JNK, BCL-2, BAX, p-53 and p21 (Cell Signaling) and actin (Santa Cruz Biotechnology).

Techniques: Western Blot, Cell Culture

Journal: Antioxidants

Article Title: New Amides and Phosphoramidates Containing Selenium: Studies on Their Cytotoxicity and Antioxidant Activities in Breast Cancer

doi: 10.3390/antiox10040590

Figure Lengend Snippet: Western blot analysis of autophagy markers SQSTM1/p62, Beclin-1, p-JNKm p-AMPK and LC3B in MCF-7 cell culture treated with I.2f. * Statistical significance with values of p < 0.05; ** statistical significance with values of p < 0.01; *** statistical significance with values of p < 0.001.

Article Snippet: Primary specific antibodies were incubated in 5% milk-TBS-Tween-20 (1 h, room temperature) to detect LC3B, Beclin-1 (D40C5), SQSTM1/p62, AMPK, JNK, BCL-2, BAX, p-53 and p21 (Cell Signaling) and actin (Santa Cruz Biotechnology).

Techniques: Western Blot, Cell Culture

Journal: Cell Research

Article Title: Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress

doi: 10.1038/s41422-019-0163-6

Figure Lengend Snippet: a Low glucose exclusively activates the lysosomal pool of AMPK in MEFs. MEFs were grown in full medium and then switched to DMEM containing reduced concentrations of glucose for 2 h, or to DMEM lacking both glucose and glutamine (starvation for glucose plus glutamine, GS + QS). Cytosolic, lysosomal, and mitochondrial fractions were prepared following the methods described in “Materials and Methods”. Fractions were then subjected to analysis of p-AMPKα and p-ACC by immunoblotting using the indicated antibodies, followed by densitometry analysis. Statistical analysis results were shown in mean ± SD; ***p < 0.001, *p < 0.05, N.S., not significant by ANOVA, n = 3. b AMP:ATP and ADP:ATP ratios are not changed in MEFs in low glucose. Adenylate nucleotide ratios in MEFs treated as in a were measured by CE-MS. Results are mean ± SD; N.S., not significant by ANOVA, n = 3. c Starvation-induced AMPK activation in liver takes place on lysosome. Mice were fed ad libitum or starved for 16 h, followed by fractionation of cytosol, lysosomes and mitochondria from liver homogenates, and subsequent immunoblotting using the indicated antibodies. d AMP/ATP and ADP/ATP ratios are unchanged in the liver of starved mice. Mice were fed or starved as in (c), freeze-clamped liver samples prepared, and AMP/ATP and ADP/ATP ratios measured by CE–MS. Results are mean ± SD, n = 6; N.S., not significant by Student’s t-test. e, f ACC1, but not ACC2, is phosphorylated in MEFs starved for glucose or in the liver of starved mice. MEFs were glucose starved or severely starved as in a, while mice were fed or starved as in c. Endogenous ACC1 and ACC2 in MEFs (e) or mice livers (f) were individually immunoprecipitated, followed by immunoblotting. Statistical analysis data of experiments in e were shown in mean ± SD; ***p < 0.001, N.S., not significant by ANOVA, n = 3. g Phosphorylation of SREBP1c, TSC2, Raptor, HDAC4, ULK1, Beclin-1, and TBC1D1, but not MFF, is observed in low glucose. HEK293T cells or MEFs were incubated in DMEM medium with (25 mM) or without glucose for 2 h, followed by analysis of phosphorylation levels of AMPK substrates as indicated. Statistical analysis data were shown in mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, by ANOVA, n = 3. h AXIN1 and AXIN2 are functionally equivalent in the lysosomal pathway of AMPK activation in HEK293T cells. AXIN2−/− HEK293T cells and its wildtype control were infected with lentivirus expressing siRNA against AXIN1. Cells were starved for glucose for 2 h and then lyzed, followed by immunoblotting. i Re-introduction of AXIN2 into AXIN1−/− MEFs restores glucose starvation-induced AMPK activation. AXIN1−/− MEFs (and its wildtype control) were infected with lentivirus expressing HA-tagged AXIN2. Cells were then starved for glucose for 2 h, followed by immunoblotting. Experiments in this figure were performed three times, except d and i twice. See also Supplementary information, Fig. S1

Article Snippet: Rabbit anti-phospho-AMPKα-T172 (cat. #2535, 1:1000 for IB), anti-AMPKα (cat. #2532, 1:1000 for IB), anti-phospho-ACC-Ser79 (cat. #3661, 1:1000 for IB), anti-ACC (cat. #3662, 1:1000 for IB), anti-LKB1 (cat. #3047, 1:1000 for IB), anti-AMPKβ1/2 (cat. #4150, 1:1000 for IB), anti-AMPKγ1 (cat. #4187, 1:1000 for IB), anti-AMPKγ2 (cat. #2536, 1:1000 for IB), anti-AMPKγ3 (cat. #2550, 1:1000 for IB), anti-AXIN1 (cat. #2074, 1:1000 for IB), anti-AXIN2 (cat. #2151, 1:1000 for IB), anti-ACC1 (cat. #4190, 1:1000 for IB and 1:100 for IP), anti-ACC2 (cat. #8578, 1:1000 for IB and 1:25 for IP), anti-phospho-SREBP1c-S372 (cat. #9874, 1:500 for IB), anti-phospho-Raptor-S792 (cat. #2083, 1:1000 for IB), anti-Raptor (cat. #2280, 1:1000 for IB), anti-phospho-TSC2-S1387 (cat. #2280, 1:1000 for IB), anti-TSC2 (cat. #4308, 1:1000 for IB), anti-phospho-HDAC4-S246 (cat. #3443, 1:1000 for IB), anti-HDAC4 (cat. #7628, 1:1000 for IB), anti-phospho-MFF-S146 (cat. #49281, 1:500 for IB), anti-MFF (cat. #86668, 1:1000 for IB), andi-phospho-TBC1D1-S660 (cat. #6928, 1:500 for IB), anti-TBC1D1 (cat. #4629, 1:1000 for IB), anti-phospho-ULK1-S555 (cat. #5869, 1:1000 for IB), anti-phospho-Beclin-1-S93 (cat. #14717, 1:1000 for IB), anti-Beclin-1 (cat. #3495, 1:1000 for IB), anti-COXIV (cat. #4850, 1:1000 for IB and 1:200 for IF), anti-PDI (cat. #3501, 1:1000 for IB), anti-Lamin B1 (cat. #13435, 1:1000 for IB), anti-β-tubulin (cat. #2128, 1:1000 for IB), anti-HA-tag (cat. #3724, 1:200 for IF), mouse anti-Myc-tag (cat. #2276, 1:1000 for IB), and HRP-conjugated mouse anti-rabbit IgG (conformation specific, cat. #5127, 1:2000 for IB) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Activation Assay, Fractionation, Immunoprecipitation, Incubation, Infection, Expressing

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: PM2.5 exposure induced autophagy in human bronchial epithelial cells. (A and B) Beas-2B cells were left untreated or were treated with PM2.5 as described in Fig. 1A and 1E. Then, the expression levels of MAP1LC3B, BECN1 and SQSTM1 were examined. (C) Beas-2B cells were left untreated or were treated with PM2.5 (100 μg/mL) for 24 h; then, autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent. (D) Beas-2B cells were treated with PM2.5 and were stained with Cyto-ID Autophagy Detection Reagent as described in (C). Then, the cells were collected and subjected to a flow cytometric analysis to quantitatively measure the autophagic fluorescence intensity inside the cells (**, P < 0.01). (E) TEM of Beas-2B cells untreated (left panel) or treated with 100 μg/mL of PM2.5 for 24 h (middle and right panels). The right panel shows a high-magnification image of the indicated region in the middle panel. Red arrows indicate double-membrane autophagic vesicles. Blue arrows indicate the particles taken up by the cells. (F) Beas-2B cells were treated with PM2.5 (100 μg/mL) alone or in combination with BafA1 (0.1 μM) during the final 4 h before the cells were harvested. Then, the expression levels of MAP1LC3B and SQSTM1 were examined 24 h after PM2.5 exposure. (G) Beas-2B cells were treated as described in Fig. 1F, and the expression levels of MAP1LC3B, BECN1 and SQSTM1 were measured. (H) Beas-2B cells were treated as described in Fig. 1F, and autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent. (I) Primary human bronchial epithelial cells were left untreated or were treated with PM2.5 (20 μg/mL) for 24 h. Then, the expression levels of MAP1LC3B, BECN1 and SQSTM1 were measured. (J) Primary human bronchial epithelial cells were treated as described in (I) and autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent.

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, Confocal Microscopy, Staining, Fluorescence

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: 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 μg/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 activity 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 μg/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 μg/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-dependent 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 promoter-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, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent 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: 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 induction 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 μg/mL) 36 h after transfection. The activation status of TP53 and the expression levels of DRAM1, BECN1 MAP1LC3B were examined 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

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: 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 μg/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 μg/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 μg/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, 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: 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 μg/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 μg/mL) 36 h after transfection. The activation status of CHEK1 and TP53 and the expression levels of DRAM1, BECN1 and MAP1LC3B were examined 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 μg/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 μg/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, Expressing, Stable Transfection, Luciferase, Activity Assay