Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 1 PBQC up regulated Nrf2 activity. (A) PBQC molecular structure is shown. (B) HeLa cells were stably transfected with ARE–luciferase reporter gene, and incubated Luci–HeLa cells with 0.1% DMSO (control) or PBQC at 1, 5, 10 mM for 6, 12, 24 and 48 h. The cell viability was analyzed by SRB assay and Luci–HeLa cells were lysed and luciferase activities were measured, setting the control group activity to 1. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. control, n ¼ 3).

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Activity Assay, Stable Transfection, Transfection, Luciferase, Incubation, Control, Sulforhodamine B Assay

Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 3 PBQC up regulated Nrf2 in HeLa cells. (A) Western blot analysis showed that Nrf2 level was up-regulated by PBQC prominently at 12 h and 24 h. (B) 100 nM si-Nrf2 dramatically decreased Nrf2 protein. And 10 mM PBQC prevented the decline of Nrf2. Set the control group activity to 1. (*p < 0.05, vs. control, n ¼ 3).

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Western Blot, Control, Activity Assay

Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 5 PBQC promoted Keap-1 glutathionylation and Nrf2 nuclear translocation. (A) WB analysis of co-IP of Keap-1 with glutathione antibody in HeLa cells treated with PBQC (0.1 and 10 mM) with 1% CS for 1, 3 and 6 h, quantification of co-immunoprecipitated Keap-1 levels. (B) Immu- nofluorescence assay of Nrf2 in HeLa cells incubated with PBQC (10 mM) for 3, 6, 12 and 24 h, nuclei were labeled with PI. Bar as present 50 mM. (*p < 0.05, **p < 0.01, vs. control, n ¼ 3).

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Translocation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, Labeling, Control

Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 6 PBQC up regulated the expressions of anti-oxidant genes and decreased the intracellular ROS level. (A and B) RT-PCR analysis of mRNA levels of Nrf2, HO-1 and GCLC treated with PBQC (1, 5 and 10 mM) PBQC for indicated times. (C) Incubated HeLa cells with 0.1% DMSO (control) or PBQC at 1, 5 and 10 mM for 1, 6, 12, 18 and 24 h, then used 5 mM DCHF probe treated all cells for 30 min. The fluorescence intensity was observed by inverted fluorescence microscope (200) and the exciting light of DCHF probe was blue light. ROS expression level was quantified by GraphPad Prism 5 software. Bar as present 22 mM. (*p < 0.05, **p < 0.01, vs. control, n ¼ 3).

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Reverse Transcription Polymerase Chain Reaction, Incubation, Control, Microscopy, Expressing, Software

Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 7 The varying degree of Nrf2 activation had different effects on the downstream Bcl-2 and Bax gene expressions. (A) RT-PCR analysis of mRNA levels of Nrf2, HO-1, GCLC, Bcl-2 and Bax in HeLa cells treated with PBQC (0.1 mM) for indicated times. (B) RT-PCR analysis of mRNA levels of Bcl-2 and Bax in HeLa cells treated with PBQC at 1, 5 and 10 mM for 1, 3, 6, 12 and 24 h, set the control group activity to 1. (#p > 0.05, *p < 0.05, **p < 0.01, vs. control, n ¼ 3).

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay

Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 8 Significant activation of Nrf2 by PBQC promoted NQO1 and p53 expressions. (A) RT-PCR analysis of mRNA levels of NQO1 in HeLa cells treated with PBQC at 1, 5 and 10 mM for 1, 3, 6, 12 and 24 h. (B) Western blot analysis of the protein level of p53, Bcl-2, Bax and b-actin as a normalization control and quantitative statistics (C and D). HeLa cells were treated with 0.1% DMSO (control) or PBQC at 1, 5 and 10 mM for 3, 6, 12 and 24 h. We set the control group activity to 1. (*p < 0.05, **p < 0.01, vs. control, n ¼ 3).

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Activity Assay

Journal: RSC Advances

Article Title: A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1S-glutathionylation and inducing apoptosis

doi: 10.1039/c7ra11935f

Figure Lengend Snippet: Fig. 10 Schematic presentation of PBQC activating Nrf2 and inducing apoptosis. (A) PBQC treatment increases Keap-1 S-glutathionylation and promotes Nrf2 activity. Subsequently, the activated Nrf2 gets into the nucleus, anti-oxidative signaling pathway is activated. The significant activation of Nrf2 by PBQC up-regulates NQO1 and p53 and specifically inhibits the increase of Bcl-2 expression. The pro-apoptotic protein Bax is significantly increased. As a result, the cancer cells undergo apoptosis.

Article Snippet: Subsequently, the membrane was probed with PARP antibody (1 : 2000) (Proteintech, USA), Nrf2 antibody (1 : 2000) (Proteintech, USA), p53 antibody (1 : 2000) (cell signaling, USA), Bcl-2 antibody (1 : 2000) (Proteintech, USA), cleaved caspase-3 antibody (1 : 2000) (cell signaling, USA), Bax antibody (1 : 2000) (Proteintech, USA) or anti-b-actin mouse monoclonal antibody (1 : 2000) (SIGMA, USA) overnight at 4 C, then was washed twice with TBST, each time for 5 min.

Techniques: Activity Assay, Activation Assay, Expressing