Supplemental Material - Various phenotypes of <i>LRBA</i> gene with compound heterozygous variation: A case series report of pediatric cytopenia patients

Supplemental Material for Various phenotypes of LRBA gene with compound heterozygous variation: A case series report of pediatric cytopenia patients by Jiafeng Yao, Hao Gu, Wenjun Mou, Zhenping Chen, Jie Ma, Honghao Ma, Nan Li, Rui Zhang, Tianyou Wang, Jin Jiang and Runhui Wu in International Journal of Immunopathology and Pharmacology</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

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

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

Table_2_Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.docx

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.</p

DataSheet_1_Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.docx

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.</p

Table_1_Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.docx

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.</p