Figure 1.
Lentiviral transduction of C/EBPβ shRNA in SUDHL-1 cell line and gene expression profiling.
(A) Western Blot analysis of the different C/EBPβ isoforms (liver-enriched activation protein (LAP*, LAP), liver-enriched inhibitory protein (LIP) in the transduced SUDHL-1 cells three days after infection demonstrates successful knockdown. Each lane contains 40 µg protein extract. α-Tubulin was used as loading control. (B) RT-qPCR analysis of C/EBPβ mRNA in the transduced SUDHL-1 cells three days after infection. Values were normalized to TBP and data were analyzed according to the 2−ΔΔCp method. Results are depicted as mRNA amount relative to untreated SUDHL-1 cells. Error bars indicate SD (n = 4). (C) Proliferation curves of the controls and C/EBPβ-shRNA infected SUDHL-1 cells are depicted up to 6 days after infection. Error bars indicate SD (n = 3). (D) C/EBPβ signature of SUDHL-1 and KiJK cells derived by C/EBPβ-shRNA. Heatmaps show the expression pattern of the 114 genes which were significantly (FDR<10%) regulated after C/EBPβ knockdown in both cell lines, SUDHL-1 and KiJK, depicting transduced cells with C/EBPβ-shRNA and controls in triplicates. The 23 candidate genes whose regulation was validated by RT-qPCR are designated. The coloured log2 scale bar represents relative gene expression changes. (E) GO term enrichment analysis was used to assess the biological functions of the 114 differentially expressed genes [23]. Selected terms (p<0.01) and the number of genes associated with them are shown. SUDHL-1 = uninfected cells, pF = empty virus, pF-C/EBPβ = virus containing the C/EBPβ shRNA sequence.
Figure 2.
Expression and ChIP analysis of selected candidate genes in ALK+ ALCL cell lines.
(A) RT-qPCR analysis of C/EBPβ, DDX21, BCL2A1, G0S2 and TRIB1 in SUDHL-1, KiJK and Karpas 299 cell lines. Values were normalized to TBP and data were analyzed according to the 2−ΔΔCp method. Results are depicted as mRNA amount relative to the C/EBPβ mRNA expression level in SUDHL-1 cells. (B) C/EBPβ, DDX21, BCL2A1, G0S2 and TRIB1 expression in SUDHL-1, KiJK and Karpas 299 detected by Western Blot. Each lane contains 40 µg protein extract. (C) Western Blot analysis of C/EBPβ, DDX21 and BCL2A1 in KiJK cell transduced with C/EBPβ shRNA four days after infection. 20 µg cytoplasmic extracts were loaded to detect G0S2 and BCL2A1 and 8 µg nuclear protein extracts were loaded to detect C/EBPβ, DDX21 and TRIB1. α-Tubulin was used as loading control. (D) Representative results of qPCR analyses to quantify the relative amounts of DNA obtained by ChIP for the potential regulatory sites of BCL2A1, TRIB1, G0S2, S100A9, DDIT4, TM4SF1, CDKN2A, PTPRC, DDX21, JUN, CCL20. DNA quantities (expressed as percentages of input) were compared for specific IPs versus IgG IP samples. One representative qPCR result of the different sites of each gene is shown. Error bars indicate SD (n = 3), p<0.05.
Table 1.
Genes differentially expressed after C/EBPβ knockdown.
Figure 3.
ChIP analysis of DDX21 gene, luciferase reporter assay and overexpression of C/EBPβ.
(A) qPCR analysis to quantify the relative amounts of DNA obtained by ChIP for the potential regulatory sites of DDX21. DDX21_I1 depicts the regulatory site in the first intron of the DDX21 gene. DNA quantities (expressed as percentages of input) were compared for specific IPs versus IgG IP samples. One representative qPCR result of the different sites is shown. Error bars indicate SD (n = 3), p<0.05. (B) Luciferase reporter assays after transfection of HEK293T cells with the reporter plasmid containing the regulatory site of the intron region of DDX21 and expression vector to overexpress the C/EBPβ isoforms LAP* and LAP. Bars represent relative luciferase activity for the respective vectors. Error bars indicate SD (n = 3), p<0.05. (C) Flow cytometric analysis of transduced Mac-1 cells and untreated controls three days after lentiviral infection. The percentage of GFP-positive cells represents the percentage of infected cells. (D) Western Blot analysis of the C/EBPβ isoforms LAP* and LAP and of DDX21 in the transduced Mac-1 and SR786 cells three days after infection. Each lane of the Western Blot contains 20 µg protein extract. Tubulin was used as loading control. Mac-1/SR786 = uninfected cells, pRRL = empty virus, pRRL-LAP* = virus containing the C/EBPβ isoform LAP* sequence, pRRL-LAP = virus containing the C/EBPβ isoform LAP sequence.
Figure 4.
Lentiviral transduction of BCL2A1-shRNA in SUDHL-1 cells and its influence on cell proliferation and apoptosis.
(A) Flow cytometric analysis of transduced SUDHL-1 cells and untreated controls three days after infection. The percentage of GFP-positive cells represents the percentage of infected cells. (B) RT-qPCR analysis of BCL2A1 mRNA in the transduced SUDHL-1 cells three days after infection. Values were normalized to TBP and data were analyzed according to the 2−ΔΔCp method. Results are depicted as mRNA amount relative to untreated SUDHL-1 cells. Error bars indicate SD (n = 3). (C) Proliferation curves of the controls and BCL2A1-shRNA infected SUDHL-1 cells up to 5 days after infection. Error bars indicate SD (n = 3). (D) Annexin V/propidium iodide staining of the controls and the BCL2A1-shRNA transduced SUDHL-1 cells three days after infection. SUDHL-1 = uninfected cells, pG-NS = pGIPZ vector with non-silencing shRNA, pG-BCL2A1 = virus containing the pGIPZ vector and the BCL2A1-shRNA sequence, PI = propidium iodide. (E) BCL2A1 immunostaining in ALCL. Representative ALK+ (left) and ALK- (middle) ALCL cases showing both a similar cytoplasmic BCL2A1 positivity in the tumor cells comparable to the expression in SUDHL-1 cells. (Immunoperoxidase, 400x).
Figure 5.
Influence of G0S2 knockdown on cell proliferation, apoptosis and cell cycle.
Validation in primary cases. (A) Flow cytometric analysis of transduced SUDHL-1 cells and untreated controls four days after infection. The percentage of GFP-positive cells represents the percentage of infected cells. (B) RT-qPCR analysis of G0S2 mRNA in the transduced SUDHL-1 cells four days after infection. Values were normalized to TBP and data were analyzed according to the 2-ΔΔCp method. Results are depicted as mRNA amount relative to untreated SUDHL-1 cells. Error bars indicate SD (n = 3). (C) Proliferation curves of the controls and G0S2-shRNA infected SUDHL-1 cells up to 5 days after infection. Error bars indicate SD (n = 3). (D) Annexin V/propidium iodide staining of the controls and the G0S2-shRNA transduced SUDHL-1 cells four days after infection. (E) Cell cycle distribution of the control and G0S2-shRNA infected SUDHL-1 cells four days after infection. The differences between the uninfected control and the G0S2-shRNA-infected cells are given in percentage in the table. SUDHL-1 = uninfected cells, pG-NS = pGIPZ vector with non-silencing shRNA, pG-G0S2 = virus containing the pGIPZ vector and the G0S2-shRNA sequence, PI = propidium iodide. (F) Western Blot analysis of caspase 3 and cleaved caspase 3 in G0S2-shRNA transduced SUDHL-1 cells four days after infection. Each lane contains 30 µg cytoplasmic extract. 15 µg protein extract of Bortezomib-treated SUDHL-1 cells were used as control. α-Tubulin was used as loading control. (G) Box plot of G0S2 expression levels obtained by RT-qPCR analysis in 6 ALK+, 8 ALK- and 4 normal lymph node samples.
Figure 6.
Influence of DDX21 knockdown on proliferation, apoptosis and pre-rRNA processing.
DDX21 expression in primary cases. (A) Flow cytometric analysis of transduced SUDHL-1 cells and untreated controls three days after infection. The percentage of GFP-positive cells represents the percentage of infected cells. (B) Analysis of DDX21 mRNA and protein downregulation, investigated by RT-qPCR analysis of DDX21 mRNA in the transduced SUDHL-1 cells three and four days after infection (upper part) and Western Blot analysis of C/EBPβ in the transduced SUDHL-1 cells four days after infection (lower part). RT-qPCR values were normalized to TBP and data were analyzed according to the 2−ΔΔCp method. Results are depicted as mRNA amount relative to untreated SUDHL-1 cells. Error bars indicate SD (n = 3). Each lane of the Western Blot contains 30 µg protein extract. Tubulin was used as loading control. (C) Proliferation curves of the controls and DDX21-shRNA double-infected SUDHL-1 cells are depicted up to 7 days after infection. Error bars indicate SD. SUDHL-1 = uninfected cells, pF = empty virus, pF-DDX21 = virus containing the DDX21 shRNA sequence. (D) Annexin V/propidium iodide staining of the controls and the DDX21-shRNA transduced SUDHL-1 cells four days after infection. PI = propidium iodide. (E) Autoradiogram of rRNA pulse-labelled with [32P] orthophosphate and chased for the indicated time. SUDHL-1 cells transduced with DDX21-shRNA, with the empty virus and untreated control cells were compared. The levels of 28S and 18S rRNA relative to those of the samples of the untreated control cells are shown at the bottom. SUDHL-1 = uninfected cells, pF = empty virus, pF-DDX21 = virus containing the DDX21-shRNA sequence. (F) ALK, CD30, C/EBPβ and DDX21 immunostaining in ALCL. The ALCL primary case (upper panel), shows nuclear and cytoplasmic ALK expression, strong nuclear positivity for C/EBPβ and strong nucleolar positivity for DDX21 in most tumor cells. Insert: Higher magnification demonstrating the strong DDX21 nucleolar positivity of the tumor cells (red arrow), in contrast to the small reactive lymphocytes with normal nucleolar expression (blue arrow). The ALK-negative case (lower panel) is negative for C/EBPβ, with reactive histiocytes serving as internal positive control. DDX21 shows only weak nucleolar positivity. Immunoperoxidase, 400x (insert, 630x).
Figure 7.
Pathway analysis of genes regulated by C/EBPβ.
Gene-gene connections (orange arrows) were analyzed by Ingenuity Pathway software and associated biological functions are shown. Genes depicted in green (red) are down (up)-regulated upon C/EBPβ knockdown in our microarray dataset. Shown are the nucleus, intra- and extracellular space and part of the cell membrane (Figure was designed with Ingenuity IPA software).