American Society of Hematology

Figure 6

$Figure 6. NPI-0052 inhibits TNF-induced p65 nuclear translocation. (A) KBM-5 cells were incubated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic and nuclear extracts were prepared, fractionated on SDS-PAGE, and electrotransferred to a nitrocellulose membrane. The analysis was performed using p65 antibodies. (B) Immunocytochemical analysis of TNF-induced p65 nuclear translocation. KBM-5 cells were incubated with 50 nM NPI-0052 for 4 hours, treated with 1 nM TNF for 15 minutes, and then subjected to immunocytochemical analysis as described in “Immunolocalization of NF-κB p65.” Cells plated with Mounting Media (Sigma-Aldrich) were analyzed under a fluorescence microscope (Lapshot-2, Nikon) equipped with a CFWN 10 /1.5 NA oil-immersion objective lens and a Photometrics Coolsnap CF color camera (Nikon). Images were acquired with MetaMorph 4.6.5 software (Universal Imaging). (C) The wild-type and p65−/− (105/mL) cells were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 1 nM TNF for 16 hours. Cell death was determined by the calcein-AM based live/dead assay as described in “Live and dead assay.” Data are for a representative experiment of 3 independent ones showing similar results. Error bars represent SD of triplicate values.$

NPI-0052 inhibits TNF-induced p65 nuclear translocation. (A) KBM-5 cells were incubated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic and nuclear extracts were prepared, fractionated on SDS-PAGE, and electrotransferred to a nitrocellulose membrane. The analysis was performed using p65 antibodies. (B) Immunocytochemical analysis of TNF-induced p65 nuclear translocation. KBM-5 cells were incubated with 50 nM NPI-0052 for 4 hours, treated with 1 nM TNF for 15 minutes, and then subjected to immunocytochemical analysis as described in “Immunolocalization of NF-κB p65.” Cells plated with Mounting Media (Sigma-Aldrich) were analyzed under a fluorescence microscope (Lapshot-2, Nikon) equipped with a CFWN 10 /1.5 NA oil-immersion objective lens and a Photometrics Coolsnap CF color camera (Nikon). Images were acquired with MetaMorph 4.6.5 software (Universal Imaging). (C) The wild-type and p65^−/− (10⁵/mL) cells were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 1 nM TNF for 16 hours. Cell death was determined by the calcein-AM based live/dead assay as described in “Live and dead assay.” Data are for a representative experiment of 3 independent ones showing similar results. Error bars represent SD of triplicate values.

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