TIF1β expression during macrophage differentiation. (A) Northern blot of TIF1β mRNA in differentiating HL-60, U937, and human peripheral blood monocytes (PBMs). HL-60 cells were induced with PMA (lanes 2–6) or DMSO (lanes 7–11). U937 cells were induced with PMA (lanes 12–17). Human PBMs were induced with GM-CSF and M-CSF (lanes 20–21). (B) TIF1β mRNA is not affected by cycloheximide. Northern analysis was performed on untreated U937 cells untreated (lane 1), treated for 4 h with PMA (lane 2) and treated with PMA and cycloheximide for 4 h (lane 3). (C) Immunoblots of extracts from differentiating U937 cells using antibodies specific for TIF1β (top) and β-actin (bottom). (D) Expression of TIF1β antisense-hammerhead ribozymes ablates TIF1β mRNA levels. U937 cells were stably transfected with plasmids expressing β-gal or TIF1β antisense-ribozymes and induced with PMA for 24 h. Four hygromicin-resistant cell clones were isolated and analyzed for expression of the mRNAs encoding antisense-ribozymes (top), endogenous TIF1β (middle), and GAPDH (bottom).

(A) Cell cycle analysis of ribozyme-expressing U937 cell lines. BrdU/PI FACS analysis of representative β-gal and TIF1β antisense-ribozyme stable cell lines. Untreated (left column) and 24 h PMA-treated (right column) cells of each clone were analyzed. Propidium iodide is on the horizontal axis, and BrdU incorporation is on the vertical axis. (B) Expression of macrophage cell surface proteins in stable U937 cell lines. Individual stable transfected clones expressing β-gal or TIF1β antisense-ribozymes were analyzed by FACS for expression of Mac1 (CD11b–CD18 heterodimer), CD11c, and CD14. Unstained cells (dashed line), untreated (normal line), and 24 h PMA-treated cells (bold line).

(A) Susceptibility of stable U937 cell lines to infection and killing by L. pneumophila. PMA-differentiated U937 stable cell clones were coincubated with the indicated number of L. pneumophila bacteria for 4 d, and then assayed for cell survival by the ability to reduce MTT. Each point represents three independent results. (B) Measurement of L. pneumophila uptake by stable U937 cell lines. The ability of U937 stable cell lines to phagocytose L. pneumophila was determined by assaying for the number of viable intracellular L. pneumophila bacteria after a 2 h coincubation of PMA-treated U937 stable cell lines with L. pneumophila. (C) Phagocytosis of FITC-labeled S. aureus bacteria. Cell clones expressing β-gal and TIF1β antisense-ribozymes were analyzed for the ability to phagocytose FITC-labeled S. aureus bacteria. Results using untreated (normal line) and 24 h PMA-treated (bold line) are shown for each clone. (D) Chemotaxis. Shown are the number of cells/mL that have migrated through the transwell membrane towards the chemoattractant chemokine, RANTES, after 6 h.

(A) Chemokine mRNA levels. Ribonuclease protection assay (RPA) was performed using probes specific for the mRNAs encoding chemokines RANTES, IP10, MIP1β, MIP1α, MCP1, IL8, and for the control L32 and GAPDH transcripts. Total RNA samples were used from cells treated with PMA for the indicated times. (B) Cotransfection of C/EBPβ and TIF1β expression plasmids with the 8XC/EBPβ-luciferase reporter plasmid into U937 cells followed by a 24 h PMA differentiation. (C) Cotransfection of C/EBPβ and TIF1β expression plasmids with the −205 HIV–LTR-luciferase reporter plasmid in U937 cells followed by a 24 h PMA differentiation. (D) Cotransfection of C/EBPβ and TIF1β expression plasmids with −205 HIV–LTR-luciferase reporter plasmids with mutations in the two C/EBPβ binding sites (−205 HIV LTR m2m3) or in the two NF-κB binding sites (−205 HIV LTR mκB) in U937 cells followed by a 24 h PMA differentiation. The absolute luciferase values were 250,000 light units with C/EBPβ alone, and 1.5 × 10⁶ light units with both TIF1β and C/EBPβ cotransfected. (E) Transfections of U937 stable cell lines expressing β-gal and TIF1β antisense-ribozymes with increasing amounts of the 8XC/EBPβ-luciferase reporter plasmid followed by a 24 h PMA differentiation. (F) Transfections of U937 stable cell lines expressing β-gal and TIF1β antisense-ribozymes with increasing amounts of the −205 HIV–LTR-luciferase reporter plasmid followed by a 24 h PMA differentiation.

TIF1β and C/EBPβ associate. (A) Whole-cell extracts from 24 h PMA-treated U937 cells were immunoprecipitated with preimmune (PI) (lanes 1,3), anti-C/EBPβ (lane 2) or anti-TIF1β (lane 4). Western analysis was then performed using anti-TIF1β (lane 2) or anti-C/EBPβ (lane 4) antibodies. (B) Whole-cell extracts from 24 h PMA-treated U937 cells were incubated with prewashed GST-C/EBPβ (lane 1), GST-TIF1β (lane 2), or GST alone (lanes 3,4). After extensive washes, the protein complexes were subjected to SDS-PAGE and immunoblotted with anti-TIF1β antibody (lanes 1,3) or anti-C/EBPβ antibody (lanes 2,4).

Detection of C/EBPβ- and TIF1β-containing complexes in EMSA analyses. (A) Nuclear extracts were prepared from untreated and PMA-treated U937 cells and used in EMSA analyses with oligonucleotides containing C/EBPβ binding sites from the α1-acid glycoprotein (AGP) promoter and the HIV LTR. (B) The shifted complex is sequence-specific and contains C/EBPβ, TIF1β, but not C/EBPα. The protein/DNA complex is competed with excess unlabeled wild-type (lanes 2,3), but not mutated (lanes 3,4), AGP oligonucleotide. EMSAs were intentionally overexposed to reveal the supershifts. Supershifted complexes are seen using antibodies against C/EBPβ (lane 8) and TIF1β (lane 9), but not with preimmune (lanes 6,10) or an antibody against C/EBPα (lane 7). (C) The experiment was identical to that shown in (B), but used the HIV LTR oligonucleotide.

Ectopic expression of TIF1β in U937 cells. (A) Retroviral constructs used to transduce U937 cells. An IRES (internal ribosomal entry site) ensures that TIF1β and YFP are coexpressed from a bicistronic mRNA message. (B) Effect of ectopic expression of TIF1β on myeloid cell surface markers Mac1 and CD11c. U937 cells were transduced with pGC-IRES-YFP (top table) or pGC-TIF1β-IRES-YFP (bottom table) induced for 24 h with optimal and suboptimal amounts of PMA. YFP-positive cells (expressing TIF1β) were assayed for levels of Mac1 and CD11c by FACS using PE-conjugated antibodies.