![Figure 2. Treatment with ibrutinib changes MYC expression in cells sensitive and resistant to ibrutinib. (A-B) Percentage of viable cells in the indicated lymphoma cell lines treated with DMSO or 0.1 or 0.5 μM ibrutinib for 72 hours. Each cell line was analyzed in triplicate, and data are shown as a bar graph corresponding to the mean ± SD. (C) Western blot analysis of pBTK levels in GCB and ABC cells after DMSO or ibrutinib treatment (0.5 μM, 6 hours) and BCR stimulation (20 μg/mL F[ab']2 anti-human immunoglobulin M, 3 min). (D-G) Phosphoflow cytometry analysis and quantification of CD19 (Tyr 531) (D-E) and GSK3β (Ser 9) (F-G) in stimulated lymphoma cells pretreated with DMSO or ibrutinib (0.5 μM) for 6 hours, normalized to the stained, unstimulated controls. (H) MYC expression in the indicated ibrutinib-resistant cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Each cell line was analyzed in 3 biological replicates. Data have been normalized to the DMSO-treated control and are shown as a bar graph corresponding to the mean ± SD. P values were calculated using 2-tailed Student t test. Significant changes between DMSO-treated and ibrutinib-treated cells were labeled with *P ≤ .05; **P ≤ .01; ***P ≤ .001. (I) Western blot analysis of the indicated ibrutinib-resistant cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Signal quantification was performed using Image Studio Lite and normalized to the DMSO-treated control. (J) MYC expression in the indicated ibrutinib-sensitive cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Each cell line was analyzed in 3 biological replicates. Data have been normalized to the DMSO-treated control and are shown as a bar graph corresponding to the mean ± SD. P values were calculated using 2-tailed Student t test. Significant changes between DMSO-treated and ibrutinib-treated cells were labeled with *P ≤ .05; **P ≤ .01; ***P ≤ .001. (K) Western blot analysis of the indicated ibrutinib-sensitive cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Signal quantification was performed using Image Studio Lite and normalized to the DMSO-treated control. (L) Western blot analysis of MYC and BTK in isogenic GCB cell lines WT or BTK KO. (M) Heat map indicating the expression levels (z-scores) of 10 MYC target genes in WSU-DLCL2 cells WT or BTK KO.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/131/21/10.1182_blood-2017-10-809210/4/blood809210f2.jpeg?Expires=1736047391&Signature=C5ud4qSrAvwZ8X5EbsvAzrwQIHAymADzIqKJ8nJRYoGZU2VfL5tXnoiR9X4Qmcr~orrpZCdXochz5~f9y6RtGHiYhq7~r0568aRGhfXTiojXWVhZq7UQ3v2vTQSS0OXeIp3zqtyCH5eiWUdee793nIKQ~8AkSWzI09as31c78JAKYSGozaX2QbaoLDbexPO-bMXtGOUm-Pal9X8SGMNeRmDv3wMAMzkDgHhfzUKXYf3XLAGc9OIerNCI6ZP6GcsgQietj1P652pdxePdkLwMQZ080DO2aNUFvgRMYhcDLdTZBv2voUysE2iPHnRA3cbA521-LDlZVWIvOxEx0qmGSA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Treatment with ibrutinib changes MYC expression in cells sensitive and resistant to ibrutinib. (A-B) Percentage of viable cells in the indicated lymphoma cell lines treated with DMSO or 0.1 or 0.5 μM ibrutinib for 72 hours. Each cell line was analyzed in triplicate, and data are shown as a bar graph corresponding to the mean ± SD. (C) Western blot analysis of pBTK levels in GCB and ABC cells after DMSO or ibrutinib treatment (0.5 μM, 6 hours) and BCR stimulation (20 μg/mL F[ab']2 anti-human immunoglobulin M, 3 min). (D-G) Phosphoflow cytometry analysis and quantification of CD19 (Tyr 531) (D-E) and GSK3β (Ser 9) (F-G) in stimulated lymphoma cells pretreated with DMSO or ibrutinib (0.5 μM) for 6 hours, normalized to the stained, unstimulated controls. (H) MYC expression in the indicated ibrutinib-resistant cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Each cell line was analyzed in 3 biological replicates. Data have been normalized to the DMSO-treated control and are shown as a bar graph corresponding to the mean ± SD. P values were calculated using 2-tailed Student t test. Significant changes between DMSO-treated and ibrutinib-treated cells were labeled with *P ≤ .05; **P ≤ .01; ***P ≤ .001. (I) Western blot analysis of the indicated ibrutinib-resistant cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Signal quantification was performed using Image Studio Lite and normalized to the DMSO-treated control. (J) MYC expression in the indicated ibrutinib-sensitive cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Each cell line was analyzed in 3 biological replicates. Data have been normalized to the DMSO-treated control and are shown as a bar graph corresponding to the mean ± SD. P values were calculated using 2-tailed Student t test. Significant changes between DMSO-treated and ibrutinib-treated cells were labeled with *P ≤ .05; **P ≤ .01; ***P ≤ .001. (K) Western blot analysis of the indicated ibrutinib-sensitive cell lines treated with DMSO or ibrutinib (0.5 μM) for 24 or 48 hours. Signal quantification was performed using Image Studio Lite and normalized to the DMSO-treated control. (L) Western blot analysis of MYC and BTK in isogenic GCB cell lines WT or BTK KO. (M) Heat map indicating the expression levels (z-scores) of 10 MYC target genes in WSU-DLCL2 cells WT or BTK KO.
