Additional file 1 of TNFα secreted by glioma associated macrophages promotes endothelial activation and resistance against anti-angiogenic therapy
Creators: name: Qingxia Wei
name: Singh, Olivia
name: Ekinci, Can
name: Jaspreet Gill
name: Li, Mira
name: Mamatjan, Yasin
name: Karimi, Shirin
name: Bunda, Severa
name: Mansouri, Sheila
name: Aldape, Kenneth
name: Zadeh, Gelareh
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1. Figure S1. RT-PCR angiogenesis array Human umbilical vein cells (HUVEC) were incubated with media alone (EBM) or conditioned medium (CM) from RAW264.7 macrophages stimulated with normal human astrocytes (Mφ-NHA) or GBM cell lines U87, U118, U251, A172 (Mφ-U87, Mφ-U118, Mφ-U251, Mφ-A172). mRNA was extracted and analyzed by an RT-PCR angiogenesis array. Results were normalized to RPL30 and ACTB. The relative quantity of significantly altered genes (&gt; 2-fold relative to Mφ-NHA or Mφ-NSC) are shown. n = 3, mean ± max/min, 95% confidence interval. Figure S2. Non-Glioma associated macrophages do not activate ECs Medium alone (EBM) or CM from normal human astrocytes (Mφ-NHA), GBM cell line (Mφ-U87), renal cell adenocarcinoma cell line (Mφ-786-O), human non-small cell carcinoma cell line (Mφ-H1299), transformed Schwanoma cell line (Mφ-Hei193) or osteosarcoma cell line (Mφ-U2OS) were analyzed by a multi-analyte inflammatory ELISA array. Only the U87 GBM cell line induces upregulation of VCAM1, ICAM1, CXCL5 and CXCL10. n = 3, mean ± max/min, 95% confidence interval. *p &lt; 0.05. Figure S3. RT-PCR angiogenesis array Human cerebral ECs (hCMEC/D3) were incubated with media alone (EBM) or conditioned medium (CM) from RAW264.7 macrophages stimulated with normal human astrocytes (Mφ-NHA) or GBM cell lines U87 (Mφ-U87). mRNA was extracted and analyzed by an RT-PCR angiogenesis array. Results were normalized to RPL30 and ACTB. The relative quantity of significantly altered genes are shown. n = 3, mean ± max/min, 95% confidence interval. Figure S4. VEGF RT-PCR assay Human umbilical vein cells (HUVEC) were incubated with media alone (EBM) or conditioned medium (CM) from RAW264.7 macrophages stimulated with normal mouse astrocytes (Mφ-NMA) or GL-261 cell lines (Mφ-GL261). mRNA was extracted and analyzed by an RT-PCR with VEGF gene. Results were normalized to RPL30 and ACTB. The relative quantity of VEGF is shown (n=3, mean ± max/min, 95% confidence interval). Figure S5. Expression of alpha 4 integrin in tumor microenvironment and on cells a) IHC with alpha 4 integrin has been performed on tumor sections from GL261 syngeneic mouse model treated with vehicle control or TNFα inhibitor, MP6-XT22. b) Flow cytometry analysis todetermine the expression of alpha 4 integrin on Raw264.7 cells and U973 cells as a positive control. Anti-Integrin alpha 4/CD49D antibody (ab202969) was diluted in 1:100 for both IHC and Flow cytometry. Alexa 488 donkey anti-rabbit secondary antibody (A32790) was diluted in 1:1000.