Additional file 1 of The HIF1α-PDGFD-PDGFRα axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin
Creators: name: Peng, Gong
name: Wang, Yin
name: Ge, Pengfei
name: Bailey, Christopher
name: Zhang, Peng
name: Zhang, Di
name: Meng, Zhaoli
name: Qi, Chong
name: Chen, Qian
name: Chen, Jingtao
name: Niu, Junqi
name: Zheng, Pan
name: Liu, Yang
name: Liu, Yan
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1. Sequencing of PCR products of genomic DNAs of HIF1A knocked out in U251 cells by HIF1A sgRNA. Figure S2. HIF1A KO in U87MG cells inhibits cell growth. A, B. HIF1A KO U87 cells grow slower than WT cells. HIF1A in U87MG cells was knocked out by Cas9-sgRNA method, which was identified by DNA sequencing of PCR product and by Western-blot (A). WT and HIF1A KO U87MG cells were seeded 1 × 104 /well in a 6-well plate and cultured for 5 days prior to crystal violet staining to observe colony growth (B). C. NSG mice implanted with HIF1A KO U87MG cells survive longer than WT U251 cells. NSG mice were intracranially implanted with HIF1A KO or WT U87MG cells (5 × 104/mouse) and Kaplan-Meier survival curves were estimated. Figure S3. HIF1α is required for the expression of PDGFD and PDGFRA under normoxia and mild hypoxia. A. The efficiency of lentiviral infection of primary Glio-1 and Glio-2. Freshly isolated cells from tumor recipients were transduced with high titer lentivirus for HIF1A-sh (mixture of HIF1A-sh1 plus HIF1A-sh2) or scrambled control (Sr-sh) by centrifugation at 2500 rpm for 2 h, and were then cultured for additional 30 h before given to mice. The GFP expression in the sh construct was photographed 24 h after transduction and represents transfection efficiency. B. Silencing efficiency of HIF1A-sh was determined by fluorescence microscopy of HEK293FT cells 24 h after co-transfection with either HIF1A-sh plus HIF1α-P2A-RFP, or Sr-sh plus HIF1α-P2A-RFP as a parallel control. C. Western blots are shown for U251 WT and HIF1A-KO cells incubated in normoxic or severe hypoxic incubator for 8 or 48 h before being lysed for Western blot. D. CoCl2 activated the promoter activities of PDGFRA and PDGFD. HEK293FT cells were transfected with PDGFRA or PDGFD promoter GFP reporter plasmids for 8 h, then treated with CoCl2 for an additional 16 h. GFP expression was then visualized by fluorescence microscopy as shown in the top panels, and quantitated by flow cytometry, summarized below. Bar graphs represent mean fluorescence intensity (MFI) readings ± SD for triplicate wells at each dose. Figure S4. HIF1α, PDGFRα and PDGF-D are frequently co-expressed in GBM. A. Co-expression of HIF1α and PDGFRα in GBM. Microarray of brain tumor with adjacent normal tissues was co-stained with primary rabbit HIF1α antibody and mouse PDGFRα antibody, and with secondary antibodies goat anti-rabbit Alexa-Fluor 594 and goat anti-mouse Alexa-Fluor 488 after washing away primary antibodies. DAPI was used to visualize nuclei (blue). B. Summary of microarray cases with double positive staining for HIF1α and PDGFRα, which was presented as percentage of total tumor subtype cases. ANB, adjacent normal brain tissue; OD, oligodendrocytoma; OA, oligoastrocytoma; AA, anaplastic astrocytoma; MB, medulloblastoma; EP, ependymoma. Data shown are representative of two independent experiments. C. Correlation of staining intensity for HIF1α with PDGFRα in GBM tissue microarray. IF staining of a 35 cases tissue microarray of GBM and 5 cancer adjacent normal cerebral tissue was performed with PDGFRα and HIF1α antibodies together as described in A. The double positive stains of cases with low, moderate, high, or very high scores were analyzed for the correlation of HIF1α with PDGFRα. D. Co-expression of HIF1α and PDGF-D in GBM. Sections of brain tumor with adjacent normal brain tissue microarray were co-stained with primary rabbit HIF1α antibody and goat PDGF-D antibody, and with secondary antibodies donkey anti-rabbit Alexa-Fluor 488 and donkey anti-goat Alexa-Fluor 594 after washing away primary antibodies. DAPI was used to visualize nuclei (blue). The correlation of staining intensity for HIF1α and PDGF-D are shown. E. Correlation of staining intensity for HIF1α with PDGFD in GBM tissue microarray. IF staining of a 38-cases tissue microarray of GBM and 6 of normal cerebrum tissues was performed with PDGF-D and HIF1α antibodies as described in D. The double positive stains of 23/38 cases with low, moderate, high, or very high scores were analyzed for the correlation of HIF1α with PDGF-D. Figure S5. TP53 mutation did not affect GBM response to Echinomycin. A. PDGF-DD levels in the medium of HIF1A KO or WT U251 cells. Levels of released PDGF-D protein were measured by ELISA. B. Echinomycin induced apoptosis of U251 cells. Annexin V staining was performed on U251 cells that were treated for 48 h with different concentrations of Echinomycin. C, D. HIF1A-KO or PDGFRA-KO cells are resistant to Echinomycin. WT, HIF1A-KO or PDGFRA-KO U251 cells were treated with different concentrations of Echinomycin for 72 h prior to determining cell viability by MTT assay. E. Primary Glio-1 cells have an R273H mutation of TP53 which is also carried by U251 cells. Sequence chromatograms are shown with arrows indicating R273H mutation. Figure S6. Echinomycin reduced the expression of HIF1α target genes. Empty vector-transfected GL261 brain tumor cells were orthotopically transplanted to recipient mice, and the mice were treated with vehicle or LEM as detailed in methods. Twenty-four hours after the final dose, the tumor cells were isolated and the cDNA was used to perform qRT-PCR. Table S1. Characteristics of Clinical Glioma Samples. Table S2. RNA-Seq data of growth factor related genes expressed in WT and HIF1α KO in U251 cells. Table S3. RNA-Seq data of metabolism related genes expressed in WT and HIF1α KO in U251 cells.