Supplemental Methods, Data 1 - 8 from Hypoxia-Induced SUMOylation of E3 Ligase HAF Determines Specific Activation of HIF2 in Clear-Cell Renal Cell Carcinoma

Supplemental methods. A, B) Sequences for the A) P117 Luc, and B) P117 HRE reporters containing the HAF binding site, the hypoxia responsive element (HRE) and the HIF ancillary sequence (HAS), with the restriction enzyme sites used for ligation into the pGL4.17 construct. C) PCR primers used in ChIP assays. Supplementary data S1. A) Quantitative TaqMan RT-PCR showing the effect of transient HAF overexpression on the transcription of HIF-2 target genes in pVHL-deficient A498 CRCC cells B) Quantitative TaqMan RT-PCR showing the effect of transient HAF overexpression on the transcription of OCT-3/4. SOX2 and NANOG in normoxia and 24hours' hypoxia in A498 CRCC cells. Supplementary data S2. A) Effects of HAF overexpression on luciferase activity of co-transfected P117 Luc or P117 containing a deletion of the HAF site (P117D), in PANC1 cells in normoxia. B) Western blot showing knockdown specificity of siRNAs targeting HIF-2α (siH2_1, siH2_2) or HAF (siHAF_1, siHAF_2) when transfected with the P117 reporter in 786-0 cells. Supplementary data S3. A) Effect of hypoxia on SUMO-2/3 modification of HAF in 786-0 cells. B) Effect of hypoxia on SUMO-1 modification of HAF in PANC-1 cells. C) Western blot showing immunoprecipitation of HIF-2α from lysates of PANC-1 cells. Supplementary data S4. A-B) Immunocytochemistry confirming specificities of A) HAF, and B) HIF-2α antibodies. Supplementary data S5. A) Growth curve of HAF overexpressing 786-0 cells in normoxia. B) Western blot showing GFP expression in suspected lesions observed in the GI tracts of mice bearing renal orthotopic 786-0 HAF or vector expressing CRCC cells. Supplementary data S6. A) Table showing multiple disease parameters of the patients represented on the CRCC TMA. B) Scatterplot showing correlation between nuclear and cytoplasmic HAF localization in CRCC TMA. Supplementary data S7. Kaplan-Meier curves showing relationship between good versus intermediate Motzer criteria in grade III/IV CRCC. Supplementary data S8. A-B) Kaplan-Meier curves showing relationships between levels of A) HIF-2α or B) HIF-1α and progression free survival (PFS) in grade III/IV CRCC.