Fig 1: Gene expression analysis of HUDEP-2 cells stably expressing SF3B1K700E. (a) Western blot analysis for SF3B1, ABCB7, and ALAS2. Relative expression level of each gene in HUDEP-2 cells stably expressing SF3B1WT or SF3B1K700E in comparison to control vector-transduced HUDEP-2 cells are described under each picture. a-Tubulin was used as a loading control. The image of each protein was cropped from the different films. The original films are presented in Supplementary Fig. S6–9 online. (b) Expression levels of ABCB7, MAP3K7 and GLRX5 were measured by quantitative RT-PCR (results shown as mean ± SD and dot plots); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (c) Western blot analysis for MAP3K7 and GATA-1. Relative expression level of each gene in HUDEP-2 cells stably expressing SF3B1WT or SF3B1K700E in comparison to control vector-transduced HUDEP-2 cells is described under each picture. a-Tubulin was used as a loading control (a). The image of each protein was cropped from the different films. The original films are presented in Supplementary Fig. S10–11 online. (d) Expression levels of MAP3K7 and GATA-1 were measured by quantitative RT-PCR (results shown as mean ± SD and dot plots); * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 2: Analysis of ABCB7-knockdown HUDEP-2 cells. (a) Expression levels of ABCB7 by quantitative RT-PCR (results shown as mean ± SD and dot plots) in undifferentiated and differentiated ABCB7-knockdown HUDEP-2 cells; * p < 0.05, ** p < 0.01. (b) Western blot analysis for ALAS2, ABCB7 and α-Tubulin in undifferentiated ABCB7-knockdown HUDEP-2 cells. Relative expression levels of ALAS2 and ABCB7 in ABCB7-knockdown HUDEP-2 cells in comparison to control shRNA-transduced HUDEP-2 cells described under the picture. α-Tubulin was used as a loading control. The image of each protein was cropped from the different fields of the film. The original film is presented in Supplementary Fig. S23-24 online. (c) Expression levels of ALAS2 by quantitative RT-PCR (results shown as mean ± SD and dot plots) in undifferentiated ABCB7-knockdown HUDEP-2 cells. (d) Representative micrograph of cytospin slides for differentiated ABCB7-knockdown HUDEP-2 cells. The upper photographs show slides stained with May–Grünwald–Giemsa stain, and the lower ones represent those stained with Prussian blue. Ring sideroblasts are indicated by black arrows. Control, shRNA5, and shRNA6 represent HUDEP-2 cells transduced with control shRNA, HUDEP-2 cells transduced with ABCB7 shRNA clone 5, and HUDEP-2 cells transduced with ABCB7 shRNA clone 6, respectively.
Fig 3: Gene expression analysis of K562 cells overexpressing SF3B1K700E. (a) Western blot analysis for FLAG, SF3B1, ABCB7, MAP3K7, ALAS2, and GATA-1. Relative expression level of each gene in K562 cells expressing SF3B1WT or SF3B1K700E in comparison to control vector-transduced K562 cells are described under each picture. a-Tubulin was used as a loading control. The image of each protein was cropped from the different fields of the film. The original film is presented in Supplementary Fig. S17–21 online. (b) Expression levels of ABCB7, MAP3K7 and GATA-1 by quantitative RT-PCR (results shown as mean ± SD and dot plots); ** p < 0.01, *** p < 0.001. (c) Comprehensive AS analysis with MISO. The graph shows the number of significant AS events detected in K562 cells stably expressing SF3B1WT or SF3B1K700E when compared with control vector-transduced K562 cells. (d, e) Read-coverage visualized by IGV around canonical 3' SS of ABCB7 exon 9 (d) and canonical 3' SS of MAP3K7 exon 5 (e). Black and red arrow indicate canonical and aberrant 3' SS, respectively. Empty, SF3B1WT and SF3B1K700E indicate K562 cells transduced with control vector, K562 cells overexpressing SF3B1WT and K562 cells overexpressing SF3B1K700E, respectively.
Fig 4: ANTXR1 silencing promotes erythroid cell differentiation in HUDEP-2 cells by interval consecutive analysis. (a) Analysis of GATA1 and ALAS2 protein expressions in HUDEP-2 cells following the knockdown of ANTXR1 the gene expression levels by western blotting and qRT-PCR. (b, c) The relative protein expression levels of GATA1 and ALAS2 were detected after HUDEP-2 cells were transfected with ANTXR1-sh5. (d, e) Quantification of western blots. Error bars represent standard deviation of three independent experiments. *P < 0.05, **P < 0.01.
Fig 5: XLSA clones show transcriptional alterations of the genes associated with glutathione synthesis. (a) Expression profiles of the genes registered to hsa04216 (ferroptosis pathway) of Kyoto Encyclopedia of Genes and Genomes in HUDEPWT, HUDEPR170L, and HUDEPR170H after 6 day differentiation with sodium ferrous citrate (SFC). The genes are arranged from the bottom in the order of the fold change of differentiated HUDEPWT to undifferentiated HUDEPWT. The heatmap is visualized using Java TreeView version 1.1.6r4 (jtreeview.sourceforge.net). (b) Western blotting for BACH1, GCLC, GCLM, and a-tubulin in HUDEP-2 clones after differentiation with SFC. Representative images of three independent experiments are presented. The cropped gel images are delineated, and the uncropped images can be found in Supplementary Fig. S9. (c, e) Chromatin immunoprecipitation with sequencing (ChIP-seq) analysis of the binding of BACH1 and MAFK for the gene region in K562 for HMOX1, GCLM, and GCLC (c); and FTH1, FTL, and SLC40A1 (e). We used ChIP-seq data from GEO (Gene Expression Omnibus) data set: BACH1 in K562, GSM935576; MAFK in K562, GSM935311; NFE2 in K562, GSM935414; and NFE2 in human erythroblasts, GSM1427076. E-blast, erythroblast. (d, f) Quantitative reverse transcriptase-polymerase chain reaction analysis for HMOX1, GCLM, and GCLC (d); and FTH1, FTL, and SLC40A1 (f) in HUDEP-2 clones after differentiation with SFC. The data were normalized relative to the GAPDH expression levels. (g) Intracellular glutathione concentration of HUDEP-2 clones after differentiation with SFC (n = 3). In (d), (f), and (g), the graphs were plotted using GraphPad Prism 9 (GraphPad Software, San Diego, CA, www.graphpad.com). The error bars represent the standard error of the mean. Each P value was calculated using Tukey’s test after a one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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