Journal: PLoS ONE

Article Title: Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib

doi: 10.1371/journal.pone.0152465

Figure Lengend Snippet: mRNA and protein level of HO-1 (a and d, respectively), GCLM (b and e, respectively) and x-CT (c and f, respectively) were measured by means of RT-PCR and Western Blotting in cell treated with 2.5 nM BTZ up to 24 h, as detailed in Materials and Methods. PCR products were separated by agarose electrophoresis and the relative intensities of the bands were normalized to 18S expression (a, b, c). The intensities of protein bands were normalized to the level of tubulin (d, e, f). The bar graphs show the mean values of three independent experiments (mean±SE). The bands show one representative experiment. In (g) tGSH and GSSG content was measured by HPLC in cells treated with 2.5 nM BTZ for 24 h. The graph shows the mean values of three independent experiments (mean±SE). In (a and d) #p<0.05 and ##p<0.01 vs 6 h BTZ-treated cells. In (b, c, d, e and g) *p<0.05 and **p<0.01 vs untreated cells.

Article Snippet: Lysates were subjected to gel electrophoresis using Mini-Protean TGX ™ Gels precast (BIO-RAD, Milan, Italy) and immunoblotted with antibodies against HO-1 (rabbit polyclonal antibody, ORIGENE, Herford,Germany), GCLM (rabbit polyclonal antibody generously provided by Dr. T.J. Kavanagh, University of Washington, Seattle, USA) [ , ], x-CT /SLC7A11 (rabbit mAb, Cell Signaling, Danvers, MA, USA) and Tubulin (mouse antibody, abcam, Cambridge, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Electrophoresis, Expressing

Journal: PLoS ONE

Article Title: Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib

doi: 10.1371/journal.pone.0152465

Figure Lengend Snippet: The number of viable cells were detected by Trypan blue dye exclusion test (a) and MTT assay (b). Cells were treated for 24 h with 2.5 nM BTZ, 1 mM BSO or silenced for HO-1, as indicated. Some samples were treated with the siRNA transfection reagent Interferin (INT) alone, as internal control. The graphs show the mean value of three independent experiments (mean±SE); *p<0.05; **p<0.01; ***p<0.001; n.s. no significant.

Article Snippet: Lysates were subjected to gel electrophoresis using Mini-Protean TGX ™ Gels precast (BIO-RAD, Milan, Italy) and immunoblotted with antibodies against HO-1 (rabbit polyclonal antibody, ORIGENE, Herford,Germany), GCLM (rabbit polyclonal antibody generously provided by Dr. T.J. Kavanagh, University of Washington, Seattle, USA) [ , ], x-CT /SLC7A11 (rabbit mAb, Cell Signaling, Danvers, MA, USA) and Tubulin (mouse antibody, abcam, Cambridge, UK).

Techniques: MTT Assay, Transfection

Journal: PLoS ONE

Article Title: Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib

doi: 10.1371/journal.pone.0152465

Figure Lengend Snippet: The binding of Nrf2 to ARE was evaluated by means of ChIP in HTLA-230 cells treated with 2.5 nM BTZ, 3 μM ATRA and 3 μM ATRA + 2.5 nM BTZ. Chromatin was immunoprecipitated with anti Nrf2 or with Normal IgG antibody, as indicated. PCRs were done with primers designed for the promoter regions of HO-1 E1 enhancer (in a and b, after 6 h or 24 h of cell treatment respectively), GCLM (in c, after 24 h) and x-CT (in d, after 24 h), as detailed in Material and Methods section. The amplification of pre-cleared DNA (input) has been done to perform the normalization of results. The graphs show the mean value of three independent experiments (mean±SE); *p<0.05 and **p<0.01 vs CTR cells; #p<0.05 vs BTZ-treated cells.

Article Snippet: Lysates were subjected to gel electrophoresis using Mini-Protean TGX ™ Gels precast (BIO-RAD, Milan, Italy) and immunoblotted with antibodies against HO-1 (rabbit polyclonal antibody, ORIGENE, Herford,Germany), GCLM (rabbit polyclonal antibody generously provided by Dr. T.J. Kavanagh, University of Washington, Seattle, USA) [ , ], x-CT /SLC7A11 (rabbit mAb, Cell Signaling, Danvers, MA, USA) and Tubulin (mouse antibody, abcam, Cambridge, UK).

Techniques: Binding Assay, Immunoprecipitation, Amplification

Journal: PLoS ONE

Article Title: Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib

doi: 10.1371/journal.pone.0152465

Figure Lengend Snippet: mRNA and protein levels of HO-1 (a and d, respectively), GCLM (b and e, respectively) and x-CT (c and f, respectively) were detected by means of RT-PCR and Western Blotting in cells treated with 2.5 nM BTZ, 3 μM ATRA and 3 μM ATRA + 2.5 nM BTZ for 24 h, as detailed in Materials and Methods. The bar graphs show the mean values of three independent experiments (mean±SE). The bands show one representative experiment. In (g) tGSH and GSSG content was measured by HPLC in cells treated with 2.5 nM BTZ and 3 μM ATRA for 24 h, as indicated, and the graph shows the mean values of three independent experiments (mean±SE); * p<0.05 and **p<0.01 vs untreated cells; #p<0.05 and ##p<0.01 vs BTZ-treated cells.

Article Snippet: Lysates were subjected to gel electrophoresis using Mini-Protean TGX ™ Gels precast (BIO-RAD, Milan, Italy) and immunoblotted with antibodies against HO-1 (rabbit polyclonal antibody, ORIGENE, Herford,Germany), GCLM (rabbit polyclonal antibody generously provided by Dr. T.J. Kavanagh, University of Washington, Seattle, USA) [ , ], x-CT /SLC7A11 (rabbit mAb, Cell Signaling, Danvers, MA, USA) and Tubulin (mouse antibody, abcam, Cambridge, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in microbiology

Article Title: Nucleoporin 85 interacts with influenza A virus PB1 and PB2 to promote its replication by facilitating nuclear import of ribonucleoprotein.

doi: 10.3389/fmicb.2022.895779

Figure Lengend Snippet: FIGURE 6 NUP85 facilitates IAV PB1-RanBP5, PB2-importin α1, and PB2-importin α7 interactions. (A) Effect of NUP85 on the interaction between RanBP5 in PB1. HEK293T cells were treated with NUP85 siRNA or NUP85 expressing plasmids and then transfected with the indicated plasmids for 24 h. Cell lysates were immunoprecipitated with an anti-Flag antibody. RanBP5 and Flag-PB1 levels were detected via Western blot. The band intensities were quantified by ImageJ, and relative precipitated HA-RanBP5 /Flag-PB1 ratios are shown at the bottom. (B–E) Interaction of PB2 with importin α family members, importin α1 (B), importin α3 (C), importin α5 (D), and importin α7 (E) in the NUP85 siRNA or NUP85 expressing plasmid treated cells. Cell lysates were immunoprecipitated with a mouse anti-Flag antibody. The bound proteins were detected by Western blot with a rabbit anti-HA antibody, a mouse anti-Flag antibody, or a rabbit anti-NUP85 antibody to detect importin α family members, NP, NUP85, and GAPDH, respectively. Images are representative of three independent experiments. The band intensities were quantified by ImageJ. The binding ratio of importin and PB1 or PB2 was calculated with the gray value of the importin divided by that of Flag, and then normalized by the that of control group, the results of which are present at the bottom of the panels. Images are representative of three independent experiments.

Article Snippet: Antibodies used for Western blot, immunoprecipitation, and indirect immunofluorescence were anti-NUP85 rabbit polyclonal antibody (catalogue No. 19370-1-AP, Proteintech, Rosemont, United States), anti-Flag mouse monoclonal antibody (catalogue No. 66008-3-Ig, Proteintech, Rosemont, United States), anti-IAV NS1 rabbit polyclonal antibody (catalogue No. GTX125990, GeneTex, CA, United States); anti-HA rabbit polyclonal antibody (catalogue No. 51064-2-AP, Proteintech, Rosemont, United States), anti-GAPDH mouse monoclonal antibody, goat anti-rabbit HRP and goat anti-mouse HRP (catalogue No. FD0063, FDR007, and FDM007, FD bio, Hangzhou, China), Tritc Conjugated goat mouse polyclonal antibody (catalogue No. HA1017, HuaBio, Hangzhou, China), anti-Histone H3 mouse monoclonal antibody (catalogue No. EM30605, HuaBio, Hangzhou, China) and Alexa Fluor 488 conjugated goat rabbit polyclonal antibody (catalogue No. HA1121, HuaBio, Hangzhou, China), Anti-Flag M2 beads (Sigma, MO, United States) and DAPI (Beyotime, Shanghai, China).

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Control