1. Volume 3, Issue 4, Pages 1153-1163 (April 2013)
C/EBPα Induces Highly Efficient Macrophage Transdifferentiation of B Lymphoma and Leukemia Cell Lines and Impairs Their Tumorigenicity 
Francesca Rapino, Eloy F. Robles, Jose A. Richter-Larrea, Eric M. Kallin, Jose A. Martinez-Climent, Thomas Graf 
Cell Reports 
Volume 3, Issue 4, Pages (April 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 1153-1163DOI: (10.1016/j.celrep.2013.03.003)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1
Activation of a Hormone-Inducible Form of C/EBPα Converts BL-Derived Cells into Macrophage-like Cells
(A) FACS plots of three Seraphina cell-derived clones expressing C/EBPαER-GFP showing GFP expression (left panel) as well as C/EBPα expression by western blot (right panel). The arrow indicates the C/EBPα band, and Ponceau staining of the proteins loaded is shown below.
(B) Mac-1 and CD19 expression of three Seraphina-derived clones 4 days after induction.
(C and D) FSC (C) correlating with size of the clones and SSC (D) correlating with granularity of the same cells shown in (A) and (B). Blue lines represent uninduced cells (0 days), red lines represent 4 day induced cells.
(E) FACS profiles of BLaER1 cells treated with E2 and stained with antibodies against Mac-1 and CD19 at the times indicated.
(F) Kinetics of differentiation antigen expression as determined from the data shown in (E).
(G) Uninduced and induced cells were fixed and stained with phalloidin Alexa Fluor 568 to detect F-actin (shown in red) as well as with DAPI to visualize the nuclei (shown in blue). Scale bar, 10 μm.
See also Figure S2.
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4. Figure 2 Gene Expression Changes during Transdifferentiation
(A) Heatmap of K-means clustering showing two groups of probes stably down- or upregulated that showed a >-4-fold change between any two samples for at least one time point of treatment.
(B) PCA of probes showed in the heatmap whose expression changes at least 4-fold during transdifferentiation, in comparison to the transcriptomes of normal B cells (blue sphere) and macrophages (red sphere).
(C and D) Kinetics of expression (exp.) of selected B cell and macrophage markers during C/EBPα-induced transdifferentiation as determined by microarray.
See also Figure S3.
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5. Figure 3
During Induced Transdifferentiation, Cells Become Phagocytic, Quiescent, and Transgene Independent
(A–D) Phagocytosis assay. Uninduced and induced BLaER1 cells were incubated with fluorescent pathogens O/N and analyzed by fluorescence microscopy and FACS. (A) The micrographs show dsRed E. coli bacteria in orange and nuclei (stained with DAPI) in blue of uninduced and induced BLaER1. (B) The FACS histograms depict red fluorescence intensity, representing overlays between uninduced cells and induced cells before (blue lines) and after bacteria incubation (red lines). (C) The micrographs show mCherry C. albicans in red, F-actin stained with phalloidin Alexa Fluor 488 (in green), and nuclei stained with DAPI in blue for uninduced and 4 day induced BLaER1 cells. (D) Experiment as in (B). Blue lines represent cells before yeast incubation and after incubation in red. Scale bars, 10 μm.
(E) Growth curves of BLaER1 cells kept with or without E2 for up to 8 days. Error bars represent the SE from three independent experiments.
(F) Change of cell-cycle status in BLaER1 cells induced by E2, as measured by BrdU incorporation and propidium iodide staining. Error bars represent the SE from three independent experiments.
(G) Transgene independence FACS profiles show changes in CD19 and Mac-1 expression in cells exposed to the inducer for different times (1, 2, and 4 days).
See also Figure S4.
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6. Figure 4 C/EBPα Impairs Tumorigenicity of BL Cells
(A, C, and D) Schemes of experimental setups (top) and Kaplan-Meier representation of animals’ median OS (bottom). If not stated otherwise, experiments were performed by injecting i.v. 2.5 × 106 BLaER1 cells into ten mice per group. Asterisks represent log rank (Mantle-Cox) test: ∗p ≤ 0.05 and ∗∗p <
(A) Effect of in vitro C/EBPα activation. Group 1 mice were injected with uninduced cells (green line). Group 2 mice received cells induced for 8 days in culture (blue line), and group 3 was as group 2, but mice were treated from 5 days before transplantation with tamoxifen in the drinking water (red line).
(B) MicroPET analysis of transplanted mice. Animals were treated with 18F-FDG and assayed to detect tumors with increased metabolic activity. The animals belonging to group 1 exhibited liver tumors after 20 days p.i., whereas the mice shown in groups 2 and 3 had no liver tumors throughout the period of examination. SUV, standardized uptake value.
(C) Effect of in vivo C/EBPα activation. All animals were transplanted with uninduced cells. Group 4 (green line) represents control mice, group 5 (blue line) was treated with tamoxifen pellets starting 5 days before transplantation, and group 6 (red line) was treated with tamoxifen pellets 10 days after transplantation.
(D) Effect of in vivo C/EBPα activation in mice transplanted with 5 × 104 uninduced BLaER1 cells. Group 9 mice were kept untreated (green line) or implanted with tamoxifen pellets 10 days after transplantation (group 10, blue line).
See also Figure S5.
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7. Figure 5 C/EBPα Impairs Tumorigenicity of Pre-B-ALL Cells
(A) FACS analysis of CD19 and Mac-1 antigen expression in RCH-ACV-tetO-CEBPα-tdTomato cells induced by doxycycline.
(B) Histogram representing the cell size and granularity of cells during transdifferentiation. Blue lines represent uninduced cells (day 0); red lines indicate induced cells (day 7).
(C) Phagocytosis assay. Micrographs show uninduced and induced (day 5) cells incubated with GFP fluorescent beads. F-actin staining is shown in red, nuclei in blue, and fluorescent beads in green. Scale bar, 10 μm.
(D and E) Schemes of experimental setups (top) and Kaplan-Meier representation of animals’ median OS (bottom). (D) Effect of in vitro transdifferentiation on tumorigenicity. Group 1 mice were injected with uninduced RCH-ACV-tetO-CEBPα-tdTomato cells (green line), group 2 with day 11-induced (tdTomatopos Mac-1pos) cells, and mice were treated with doxycycline (doxy) in the drinking water. (E) Effect of in vivo C/EBPα activation. Mice in groups 3 and 4 were injected with uninduced RCH-ACV-tetO-CEBPα-tdTomato cells and kept untreated (green line) or treated with doxycycline in the drinking water starting 5 days before injection (blue line). Asterisks represent significance in the log rank (Mantle-Cox): ∗∗p <
See also Figure S6.
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8. Figure S1
B Cell and Macrophage Gene Expression Changes in Partial Responder Cell Lines and Exhibits C/EBPα Dose Dependence, Related to Table 1
(A) Relative expression (log2) of B cell and macrophage genes detected by qRT-PCR in three partial responder cell lines (Z138, HBL2 and Namalwa) induced for different time (0h, 48h, 96h) with average of biological duplicates and error of the mean.
(B and C) FACS plots of Val and Z138 cells containing the Tet transactivator and TetO-C/EBPα-tdTomato virus, showing Tomato expression (left) and Mac-1/CD19 expression (right) in uninduced cells (0d) as well as in cells induced for 4 days with doxycycline, gating on cells expressing high, medium and low levels of Tomato.
(D) FACS plots of Daudi cell line expressing the C/EBPαER GFP construct, showing GFP expression (left) and Mac-1/CD19 expression (right) in cells induced for 4 and 8 days with E2, gating on cells expressing different levels of GFP.
(E) Representative Western blot showing the expression of C/EBPα in four uninduced (-) or 3 day induced (+) cells. The arrow indicates C/EBPα. Ponceau staining showing comparable loading of the samples is shown at the bottom.
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9. Figure S2
C/EBPα Dose Dependency of Differentiation Marker Expression as well as MYC Expression and Immunoglobulin Rearrangements in Transdifferentiated BLaER1 Cells, Related to Figure 1
(A) FACS plots of Seraphina cells infected with C/EBPαER-GFP, showing GFP expression (left) and Mac-1/ CD19 expression in uninduced cells (0d) as well as in cells induced for 4 days with E2, gating on cells expressing high, medium and low levels of GFP.
(B) Tomato levels and Mac-1/CD19 expression in a sorted pool of Seraphina cells infected with tetO-C/EBPα-tdTomato and induced for 5 or 9 days with doxycycline.
(C) Kinetics of endogenous MYC expression during transdifferentiation, showing B cells and macrophages as a control. Graph shows median of the data points, errors represent the data range as determined from Agilent microarray. Western blot (right panel) showing MYC expression in 0d and 7d induced BLaER1 cells; GFP expression level is shown as a control.
(D) Immunoglobulin heavy chain rearrangements in day 0 and day 8 induced cells detected by semi-nested PCR using consensus primers directed to FR1c, FR2 and FR3 of the VH and JH regions. GAPDH is shown as a control.
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10. Figure S3
GO Analysis of Gene Expression Changes during Transdifferentiation, and Array Validation, Related to Figure 2
(A) GO analysis of biological functions of the 2 groups of genes down- or upregulated (Figure 2A), showing the p-values of the first 5 categories.
(B) Agilent expression values (log2) of selected B cell and macrophage markers in control primary B cells and macrophages.
(C) Relative expression (log2) of genes shown in Figures 2C and 2D. Average of biological duplicates and error of the mean.
(D) CD79a and CD14 protein expression detected by FACS in uninduced (d0) and induced (d7) BLaER1 cells (red line) compared to isotype control stained cells (gray line).
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11. Figure S4
Cell-Cycle Analysis, Strategy for Induction of Transgene Independence, and Endogenous C/EBPα Upregulation, Related to Figure 3
(A) Propidium iodide staining of uninduced and induced cells at different times p.i. showing a gradual decrease in S/G2/M and absence of hypo- or hyper-diploid cells.
(B) Scheme for pulse induction experiments.
(C) FACS profiles of CD19 and Mac-1 expression in cells pulsed for 3 days followed by washout or continuously exposed to E2, showing essentially no differences.
(D) Kinetics of C/EBPα expression during induced transdifferentiation, with B cells and macrophages used as controls. Graph shows median of the data points, error bars represent the data range as determined from Agilent microarray. The values shown represent endogenous C/EBPα, which differed from the C/EBPα transgene of mouse origin.
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12. Figure S5
In Vivo Induction of C/EBPα and Analysis of Lymphoid Tumors and Tumor-Derived BLaER1 Cells, Related to Figure 4
(A) FACS plots of BLaER1 cells treated with 2 μΜ tamoxifen at different times, showing differentiation induction as monitored by Mac-1 and CD19 expression, as well as by forward and side scatter.
(B) Histopathological analysis (Haematoxylin Eosin staining, HE) of a liver from a mouse of Group #3 at 28 days after transplantation, showing discrete areas of tumor cells.
(C) HE staining of livers of mice from the three experimental groups analyzed in Figure 4B).
(D) FACS plots from the liver of the same animals as shown in B, showing CD19 and Mac-1 expression.
(E) FACS plots of BLaER1 cells and BLaER1 cells derived from the liver of an animal in group #3 (Figure 4B), after E2 induction in culture, showing reciprocal regulation of Mac-1 and CD19 expression and changes in FSC and SSC in both cell cultures.
(F) Experiment performed as in Figure 4C but mice were treated with tamoxifen in the drinking water Median OS Group 4, 29 ± 2.5 versus group 5, 32 ± 10; group #4, 29 ± 2.5 versus group 6, 31 ± 3.5. Asterisks represent long-rank (Mantle-Cox) test p = (∗∗).
(G) Effect of tamoxifen on Seraphina cells. Mice (n = 5) were injected with 2.5x106 Seraphina cells: in group #7 mice were kept untreated (green line); group #8 mice were treated with tamoxifen pellets starting 5 days before transplantation (blue line).
(H) Survival curve of mice injected with different dilution of BLaER1 cells to determine the minimum number of cells able to induce tumors.
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13. Figure S6
Analysis of Functionality and Cell-Cycling Properties of RCH-ACV-tetO-CEBPα-tdTomato-Induced Cells, Related to Figure 5
(A) Phagocytosis assay; uninduced and induced RCH-ACV-tetO-CEBPα-tdTomato cells were incubated with GFP fluorescent beads overnight and analyzed by FACS. The FACS histograms depict FITC fluorescence intensity, representing overlays between uninduced (d0) cells and induced cells (d5) before (blue lines) and after incubation (red lines) with beads.
(B) Growth curves of RCH-ACV-tetO-CEBPα-tdTomato cells kept with or without doxycycline for up to 7 days. Error bars represent the standard error from three independent experiments.
(C) Effects of doxycycline on RCH-ACV cells. Two groups of mice (n = 5) were injected with 1x105 RCH-ACV cells and left untreated (group #5, green line) or treated with doxycycline in the drinking water (group #6, blue line).
Cell Reports 2013 3, DOI: ( /j.celrep )
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