Supplemental Movie 1: Time-lapse imaging in hypoxia of a single HeLa cell ectopically expressing EGFP-HIF-2α. from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplental Movie 2:Fast imaging of a HeLa cell ectopically expressing EGFP-HIF-2α. from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplemental movie 5: Half-nucleus bleaching of a cell expressing EGFP-HIF-2α from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplemental movie 7 from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplental Movie 3: Trajectory of individual HIF-2α speckle in single cell (example 1). from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity