Additional file 1: of Targeting FLT3 in acute myeloid leukemia using ligand-based chimeric antigen receptor-engineered T cells

Figure S1. Flow cytometry analysis of CD45+CD33+ leukemia cells in peripheral blood of 14 and 7 days before death of leukemia mice. Figure S2. FLT3 SFI of three cord blood CD34+ HSCs, five FLT3+ leukemia cell lines, and leukemia cells of ten AML patients were analyzed by flow cytometry. (PNG 1277 kb

Oncogenes <i>WNT1</i>, <i>WNT2</i>, <i>c-MYC</i> and <i>NOTCH1</i> are specifically overexpressed in SP of human lung adenocarcinoma cell line A549.

<p><b>A</b>. SP cells in A549 were detected and separated by FACS using the Hoechst33342 dye efflux method (left) and verified by their failure to efflux this dye after incubation with FTC, a specific inhibitor of multidrug transporter-ABCG2 (right). This figure represents 1 of 3 experiments. The mRNA expression levels of oncogenes and stem cell genes were compared between the SP and NSP cells by using semi-quantitive RT-PCR, real-time RT-PCR in <b>B</b> and <b>C</b> separately, n = 3. <b>D</b>. Western blotting results of WNT1, WNT2, NOTCH1, c-MYC and SOX2 proteins in SP and NSP of A549 cells.</p

Silencing of <i>SOX2</i> inhibits tumorigenesis and regulates expression of oncogenes in vivo.

<p><b>A</b>. A549 cells were injected via the tail vein into NOD/SCID mice and the bioluminescence images of xenografted tumor were taken at the times indicated. <b>B</b>. Bioluminescence intensity was measured and plotted, n = 5. <b>C</b>. HE staining was used to detect xenografted tumors in lung tissues of xenografted NOD/SCID mice. <b>D</b>. Immunohistochemistry staining of SOX2, c-MYC, NOTCH1 and WNT1 (all shown in brown color) from lung tissues of xenografted NOD/SCID mice in situ. <b>E.</b> Immunofluorescence of WNT2 (green) in xenografted murine lung tissues. All microscopy images were recorded under a 40× objective. The figures in <b>C</b>, <b>D</b> and <b>E</b> represent 1 of 5 experiments. The tumor regions in <b>C</b>, <b>D</b> and <b>E</b> were all circulated by dash lines.</p

The most enriched 20 signaling pathways of SOX2 target genes in KEGG database.

<p>DEG: differentially expressed genes.</p

The primers used for RT-PCR and real-time RT-PCR.

<p>The primers used for RT-PCR and real-time RT-PCR.</p

SOX2 regulates the expression of oncogenes in both A549 and H460 cells.

<p><b>A</b>. A549-H1tetO-shRNA-SOX2 and control cells were incubated with Dox for 4 days and silencing of <i>SOX2</i> gene was confirmed by Western blotting. <b>B</b>. FACS analysis of the SP in A549 cells with or without <i>SOX2</i> silencing. The SP cells in A549 were confirmed by their ability to efflux Hoechst 33342 dye in the absence of FTC (up panel, FTC-), but fail to efflux the dye after incubation with FTC (low panel, FTC+). <b>C</b>. The percentage of SP in A549 cells from each experiment group was averaged from three independent experiments and plotted. <b>D</b>. Real-time RT-PCR was used to compare mRNA expression levels of oncogenes in A549 cell line with or without down-regulation of SOX2, n = 3. <b>E</b>. Protein expression levels of <i>WNT1</i>, <i>WNT2</i>, <i>NOTCH1</i> and <i>c-MYC</i> genes were detected in A549 and H460 cells with <i>SOX2</i> silencing by Western blotting.</p

Cluster image of the other target cancer genes of SOX2.

<p>Standardized TPM (transcripts per million clean tags) values were applied to compare the other target cancer genes expression levels between A549 cells with <i>SOX2</i> silencing (shRNA-SOX2) and their control (shRNA-Con). The transcription of each gene is represented by a square with a color that codes for the values of Lg TPM. Specifically, bright green represents low expression, bright red represents strong expression. The target genes whose transcriptions are up-regulated with the silencing of <i>SOX2</i> were presented in <b>A</b> and those down-regulated genes were presented in <b>B</b>.</p