American Society of Hematology

Figure 3.

$Figure 3. NF-κB pathway is activated in CLL and MCL cells preincubated with agonist mix. CLL and MCL patient PBMCs were preincubated with agonist mix for 12 hours. At the end of 12 hours, a second dose of agonist mix was added and incubated for another 6 hours. Cytoplasmic and nuclear localization of RelB or RelA in a minimum of 500 live/CD5+/CD19+ cells in CLL and MCL samples was analyzed in ImageStream using IDEAS ImageStream analysis software. (A-B) Representative images showing CD5 (red), CD19 (violet), RelB (green), 7AAD (orange), and 7AAD/RelB in CLL (A; top panel) and MCL (B; top panel) patient PBMCs or CD5 (red), CD19 (violet), RelA (green), 7AAD (orange), and 7AAD/RelA in CLL (A; bottom panel) and MCL (B; bottom panel) patient PBMCs. Original magnification ×60. (C-D) Percentage of nuclear localization of RelB and RelA in 16 CLL patient PBMCs (Pts 01-03, 7, 12, 21, 25-28, 33, 35, 43, 46, 51, and 52) (C) and 7 MCL patient PBMCs (Pts 23, 38, 45, 49, 55, 60, and 66) and Mino cells (D) preincubated with or without agonist mix. (E-G) CLL patient PBMCs (N = 3) were incubated with agonist mix for 12 hours and treated with BMS-345541 (16 μM) or IBR (0.1 μM) + VEN (25 nM) as well as a second dose of agonist mix for another 6 hours. The representative image of similarity score histograms showing shift in RelB (E) or RelA (F) colocalization with 7AAD. Percentage of nuclear localization of RelB and RelA in different treatment groups (G). (H-I) Mino cell line preincubated with agonist mix for 12 hours and treated with BMS-345541 (0-64 μM) and a second dose of agonist mix for another 6 hours. RelA, RelB, histone (H3), and tubulin proteins in cytoplasmic and nuclear fractions were analyzed by western blot (H). Bar graphs showing relative band densities of RelB and RelA proteins in the nuclear fractions (I). The data were average from 2 independent experiments. RelB and RelA in nuclear fractions were normalized to histone. Data are expressed as means ± SD. The statistical significance was determined by the paired Student t test. *P < .05, **P < .01, ****P < .0001.$

NF-κB pathway is activated in CLL and MCL cells preincubated with agonist mix. CLL and MCL patient PBMCs were preincubated with agonist mix for 12 hours. At the end of 12 hours, a second dose of agonist mix was added and incubated for another 6 hours. Cytoplasmic and nuclear localization of RelB or RelA in a minimum of 500 live/CD5⁺/CD19⁺ cells in CLL and MCL samples was analyzed in ImageStream using IDEAS ImageStream analysis software. (A-B) Representative images showing CD5 (red), CD19 (violet), RelB (green), 7AAD (orange), and 7AAD/RelB in CLL (A; top panel) and MCL (B; top panel) patient PBMCs or CD5 (red), CD19 (violet), RelA (green), 7AAD (orange), and 7AAD/RelA in CLL (A; bottom panel) and MCL (B; bottom panel) patient PBMCs. Original magnification ×60. (C-D) Percentage of nuclear localization of RelB and RelA in 16 CLL patient PBMCs (Pts 01-03, 7, 12, 21, 25-28, 33, 35, 43, 46, 51, and 52) (C) and 7 MCL patient PBMCs (Pts 23, 38, 45, 49, 55, 60, and 66) and Mino cells (D) preincubated with or without agonist mix. (E-G) CLL patient PBMCs (N = 3) were incubated with agonist mix for 12 hours and treated with BMS-345541 (16 μM) or IBR (0.1 μM) + VEN (25 nM) as well as a second dose of agonist mix for another 6 hours. The representative image of similarity score histograms showing shift in RelB (E) or RelA (F) colocalization with 7AAD. Percentage of nuclear localization of RelB and RelA in different treatment groups (G). (H-I) Mino cell line preincubated with agonist mix for 12 hours and treated with BMS-345541 (0-64 μM) and a second dose of agonist mix for another 6 hours. RelA, RelB, histone (H3), and tubulin proteins in cytoplasmic and nuclear fractions were analyzed by western blot (H). Bar graphs showing relative band densities of RelB and RelA proteins in the nuclear fractions (I). The data were average from 2 independent experiments. RelB and RelA in nuclear fractions were normalized to histone. Data are expressed as means ± SD. The statistical significance was determined by the paired Student t test. *P < .05, **P < .01, ****P < .0001.

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