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. Author manuscript; available in PMC: 2020 Aug 29.
Published in final edited form as: Cell Rep. 2018 Aug 21;24(8):2155–2166. doi: 10.1016/j.celrep.2018.07.055

Figure 3. Effects of BET Inhibition on PI3K-GSK3β Signaling and Chemokine Secretion in DLBCL Cells.

Figure 3.

(A) Effect of JQ1 treatment (0.5 μM for 24 hr) on the phosphorylation level of PI3K pathway proteins including GSK3β (S9) in DLBCL and BL cell line panel, as determined by a luminex multiplex assay.

(B) Western blot confirming sustained effects of JQ1 on c-MYC, GSK3β (S9) and total GSK3β levels in TMD8 cells for up to 48 hr.

(C) Confirmatory western blots showing similar effects of JQ1 and CPI-203 (1 μM, 24 hr) on p-GSK3β S9, total GSK3β, and β-catenin levels in 2 BL cell lines (RAJI and CA-46). Numbers below β-catenin blots indicate fold-change of protein expression versus DMSO (normalized to the relative loading controls) evaluated by densitometry analysis using the ImageJ software.

(D) Nuclear-cytoplasmic fractionation experiments confirming increased nuclear β-catenin levels in ABC-derived DLBCL cell lines (TMD8, HBL-1) following treatment with BET inhibitors (JQ1 or CPI-203 0.5 μM for 24 hr). Vinculin and lamin B1 were used as loading controls for cytoplasmic and nuclear fractions, respectively. Numbers indicate fold-change of nuclear β-catenin protein expression versus DMSO (normalized to the relative loading controls), evaluated by densitometry analysis, using the ImageJ software. N, nuclear protein fractions; C, cytoplasmic protein fractions.

(E) Effect of JQ1 treatment (0.5 μM for 24 hr) on cytokine-chemokine levels as measured by a multiplex assay in 9 representative DLBCL cell lines of ABC and GCB origin (SUDHL-4, SUDHL-6, SUDHL-8, DB, BJAB, TMD8, HBL-1, and U2932), and 1 BL cell line (CA-46). As shown, JQ1 upregulated the PI3K-dependent chemokines MIP-1α and MIP1β mostly in ABC and BL cell lines. In some cell lines, cytokine levels were below detection. Results are shown as average fold change value of cytokine-chemokine concentration in cell culture supernatants (versus DMSO) of 3 independent experiments.

(F) Standard ELISA confirming significant MIP-1α upregulation in ABC-derived DLBCL cell lines (TMD8, HBL-1) and BL cell lines (CA-46) after treatment with JQ1 or CPI-203 0.5 μM for 24 hr. Error bars represent SEM of triplicate experiments. Differences between groups (JQ1 or CPI versus DMSO) were calculated with the Student’s t test. *p < 0.05, **p < 0.01. BD (below detection).