EMBO/SourceData huggingface dataset:
Figure 3Western blotting of PRMT5 and DDX5 knockdowns.Representative images of S9.6 staining in siCTL, siDDX5, or siPRMT5 knockdown at transcription on (TA-KR) or off (TetR-KR) genomic loci in U2OS TRE cells. The nuclei are visualized by DAPI.Quantification of the S9.6 foci intensity in the indicated conditions. Three independent experiments were performed and 50 cells were analyzed in each experiment. Error bars represent mean of S9.6 foci intensity quantification with s.e.m. The statistical analysis was performed by two-tailed Student's t-test (Mann-Whitney test U-test). ****, p < 0.0001.
Figure 4U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.Western blotting of whole cell extracts from siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 transfected cells.U2OS cells transfected with siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.
Figure 5U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies.Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant.Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the S.E.M from 3 independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and S.E.M. from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.HEK293 cells were transfected with siCTL, or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). Forty eight hours later, all the cells were subjected to DRIP-qPCR analysis. The average with the S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and n.s.: not significant.
Figure 6HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies.Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag-DDX5 WT (1-614) or truncated Flag-DDX5 proteins (60-614; 1-554; 1-435). WCE of the transfected cells (left panel) were Western blotted with anti-Flag and anti-XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed and anti-Flag immunoprecipitations (right panel) performed for the presence of co-immunoprecipitating endogenous XRN2 by Western blotting with anti-XRN2 antibodies. M denotes molecular mass markers in kDa.Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.
Figure 8Schematic representation of the β-actin locus and the location of the genomic PCR primers used for ChIP-qPCR and DRIP-qPCR. Prom (promoter), RB (region B), poly(A) and RD (region D) indicate the regions analyzed by qPCR.HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed. The XRN2 ChIP signal was normalized to the Pol II ChIP signal. WCE were immunoblotted with anti-DDX5 and α-tubulin antibodies, the latter was used as a loading control. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.HEK293 cells were transfected with siCTL, siDDX5 or siXRN2. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.HEK293 cells were transfected with siCTL or siPRMT5. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.HEK293 cells were transfected with empty plasmid (-), wild type Flag-DDX5 (WT) or its RK mutant (RK) and subjected to western blot and Flag-DDX5 ChIP-qPCR analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001; n.s. not significant.
Figure 1(A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame.Human osteosarcoma U2OS cells (B or neuroglioma H4 cells (D stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated , or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm.Human osteosarcoma U2OS cells F) or neuroglioma H4 cells H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap (G, I).(J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).
Figure 2Hepatoma HepG2 cells were treated with the indicated concentrations of 3,4-DC for 8 h (A,B) GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in (B Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05Hepatoma HepG2 cells were treated with 30 µM 3,4-DC for the indicated time points (C,D). Then, cells were processed to measure LC3 and p62 protein levels by SDS-PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in D). Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05).(E,F) HepG2 were treated with 30 µM 3,4-DC for 8 h in the presence or absence of chloroquine (CQ, 50µM) for 4 h. SDS-PAGE and immunoblot were performed Band intensities of LC3-II and GAPDH were assessed and their ratio (LC3-II/GAPDH) was calculated in (F). Data are means ± SEM of at least three independent experiments (* = p < 0.05, ** = p < 0.01 versus untreated control; ##=p<0.01 versus CQ).(G,H) U2OS-GFP-LC3 cells were treated with 30 µM 3,4-DC, 10 µM rapamycin, or left untreated for 16 h in the presence or absence of CQ for 4 h. GFP-LC3 dots were measured. Data are means ± SD (*** = p < 0.001 versus untreated; ###=p<0.001 versus CQ).(I,J) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP-RFP proteins (GFP-RFP-LC3) were treated with the indicated raising doses of 3,4-DC or 50 µM chloroquine (CQ) for 16 h. After fixation, GFP-LC3 and RFP-LC3 dots were measured by automated image acquisition and analysis. GFP and RFP double positive LC3 dots indicating autophagosomes (GFP+), and GFP negative but RFP positive LC3 dots indicating autolysosomes (GFP-) were counted in (J). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in I. Scale bar equals 10 µm.
Figure 3A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH.(C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ).(E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).,G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm.
Figure 4U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (A). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. Representative images are shown in (B). Scale bar equals 10 µm.(C, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm.(E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page.MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm.MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels.(J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC)(M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).
Figure 5U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblotU2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm.U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblotU2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy ata are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown cale bar equals 10 µm.U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G).U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively).(I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).(J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC).
Figure 6(A-D) Female C57/BL6 animals were treated with 3,4-DC for 24 h. Following, liver and heart tissue was excised and cells were subjected to subcellular fractionation. Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 (A, C). GAPDH and H3 were used as controls for cytosolic and nuclear fractions, respectively. Band intensities of nuclear TFEB, TFE3 and H3 were measured and ratios of nuclear TFs versus H3 (TFsnuc/H3) were calculated in (B, D). Data are means ± SEM of at least three mice (*** = p < 0.001 versus Ctr)GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in heart (I, J) tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).
Figure 7(A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm.(B,C) The size of the infarction area (indicated by pale color) per area at risk (AAR) (B) and the global AAR per LV (C) were measured (mean value ±SEM, **p<0.01, #p<0.05 vs WT/Ctr).
Figure 8A. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation.(C-E). Cytofluorometric characterization of tumor infiltrating lymphocytes (TIL), in particular FOXP3+ regulatory T cells (Treg), CD8+ cytotoxic T lymphocytes and the ratio of CD8+ T cells over Treg.Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl/6 mice treated as indicatedGrowth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD (H-I) and were evolving in immunocompetent C57Bl/6 mice or immunodeficient nu/nu mice (G), treated as indicated(J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14).
Figure 1B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.
Figure 2A CFU-MK assays with CD34+ cells expressing AS-RBM15. MK colonies staining for acetylcholinesterase activity were counted (right). P value was calculated using Student's t test (n=3, mean ± SD).B The number of CD41+CD42+ mature MK cells was determined by FACS analysis. CD34+ cells expressing sh-AS-RBM15 were grown in pro-MK medium containing thrombopoietin and stem cell factor (TPO/SCF) for five days (n=4, mean ± SD).D FACS analysis of CD34+ cells grown in pro-MK medium and expressing AS-RBM15 full length or Δ5 or Δ3. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).E The percentage of CD41+CD42+ cells in CD34+ cells expressing AS-RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro-MK medium for five days. P value was calculated by t test, p<0.01, (n=3, mean ± SD).