STAT3 regulates a subset of hypoxia-dependent genes.

A–D Transcriptome analysis by RNA-seq. A: Genes that were differentially expressed (log2[fold change (FC)] > + 0.75 or <−0.75; q-value < 0.01, Benjamini–Hochberg adjustment, as indicated by dashed lines) between Stat3^+/+ cells in normoxia and Stat3^+/+ cells in hypoxia; n = 4 biological replicates. Expression levels of genes that were down- (B) and upregulated (C) in Stat3^+/+ in hypoxia relative to Stat3^+/+ cells in normoxia. Each boxplot shows the 1st, 2nd and 3rd quartiles; the whiskers show the minimum and maximum values. D heatmap of RNAseq data reporting the expression of genes that are affected by hypoxia in Stat3^+/+ cells compared to Stat3^+/+ normoxic cells, but that require STAT3 to be properly altered in their expression. E expression level of Vegfa, Hk1, Hk2, Pfkp and Hilpda in normoxic and hypoxic Stat3^+/+ and Stat3^−/− mESCs taken from RNAseq data. F Gene expression analysis by RT–qPCR of Stat3^+/+ and Stat3^−/− cells treated in normoxic and hypoxic mESCs of the same genes shown for the RNAseq. Mean ± SEM of n = 4 experiments, with each replica shown as a dot.

STAT3 physically interacts with HIF1α.

A RT-qPCR analysis of Hif1α mRNA expression on murine Stat3^+/+ and Stat3^−/− murine ESCs incubated either in low and in high oxygen tensions. B, C western blot analysis of HIF1α and STAT3 from protein extracts of Stat3^+/+ and Stat3^−/− murine ESCs incubated either in low and in high oxygen tensions. Representative pictures (B) and quantifications (C). β-Actin was used as an internal control. 3 independent biological replicates were used. D PLA with anti-STAT3 and anti-HIF1α antibodies. Scale bar = 200 μm. Quantification of dots divided for the number of nuclei detected with DAPI (blue). n = 3 independent biological replicas were used. Scale bar = 200 μm. Mean ± SEM.

Stat3 is necessary for Hif1α transcriptional activity.

A Representative pictures of HRE:mCherry reporter zebrafish treated with AG490 in combination with low oxygen tension, DMOG, and Dex from 3-6 dpf and CoCl₂ from 5-6 dpf. Scale bar 500 μm. n = 18 DMSO normoxia, 11 DMSO hypoxia, 18 AG490, 9 AG490 hypoxia, 11 Dex, 11 Dex AG490, 12 DMOG, 12 DMOG AG490, 18 CoCl₂, 12 CoCl₂ AG490 (larvae used for this experiment came from three independent breeding between wild type zebrafish). B Fluorescence quantification of HRE:mCherry reporter zebrafish treated with AG490 in combination with low oxygen tension. C Fluorescence quantification of HRE:mCherry reporter zebrafish treated with AG490 in combination with 10 μM Dex. D Fluorescence quantification of HRE:mCherry reporter zebrafish treated with AG490 in combination with 50 μM DMOG from 3–6 dpf. E Fluorescence quantification of HRE:mCherry reporter zebrafish treated with AG490 in combination with 0.1 mM CoCl₂ from 5-6 dpf. F, G Representative pictures and fluorescence quantification of HRE-mCHerry reporter zebrafish larvae treated with 12.5 μM PD98059 in combination with low oxygen tension. n = 31 DMSO normoxia, 29 PD98059 normoxia, 28 DMSO hypoxia, 29 PD98059 hypoxia (larvae used for this experiment came from three independent breeding between wild type zebrafish). Scale bar = 500 μm. H, I Representative pictures and fluorescence quantification of stat3^+/+, stat3^+/− and stat3^−/− sibling larvae in HRE:mCherry transgenic background treated with low oxygen tension from 3–6 dpf. n = 7 stat3^+/+ normoxia, 9 stat3^+/+ hypoxia, 6 stat3^+/− normoxia, 7 stat3^+/− hypoxia, 8 stat3^−/− normoxia, 6 stat3^−/− hypoxia (larvae used for this experiment came from three independent breeding between stat3^+/− zebrafish). Scale bar = 500 μm. Mean ± SEM.

Stat3 and Hif1α cooperate in the nucleus for the transcription of Hif1α target genes.

A, B Representative pictures and fluorescence quantification of 4-dpf HRE:mCherry reporter larvae treated with either DMSO or 50 μM AG490 for 24 h after injection of hif1ab DA mRNA. n = 3 independent biological replicates; 10 DMSO, 20 DMSO hif1ab DA mRNA, 19 AG490, 24 AG490 hif1ab DA mRNA (larvae used for this experiment came from three independent breeding between wild type zebrafish). Scale bar 500 μm. C RT-qPCR analysis of vegfa of 4-dpf larvae injected with hif1ab DA mRNA and treated with AG490 (n = 4 independent biological replicates composed by pool of 15 larvae, each dot represents a pool of larvae). D RT-qPCR analysis of hk1 of 4-dpf larvae injected with hif1αb DA mRNA and treated with AG490 (n = 4 independent biological replicates composed by pool of 15 larvae, each dot represents a pool of larvae). E RT-qPCR analysis of vegfa, hk1, egln3, and hif1αb on EGFP-positive and EGFP-negative cells sorted from SBE:EGFP transgenic adult intestines (three adult intestines were pooled together in two biological replicas). Mean ± SEM.

stat3 genetic ablation affects angiogenesis, and macrophage migration.

A, B Representative pictures and fluorescence quantification of the trunk of 54-hpf stat3^+/+, stat3^+/− and stat3^−/− in Tg(Fli1:EGFP)^y1 transgenic background incubated in normoxia and hypoxia for 6 h. Scale bar: 1 mm. n = 15 stat3^+/+ normoxia; 17 stat3^+/+ hypoxia; 21 stat3^+/− normoxia; 20 stat3^+/− hypoxia; 14 stat3^−/− normoxia; 14 stat3^−/− hypoxia (larvae used for this experiment came from three independent breeding between stat3^+/− zebrafish). C, D Representative pictures and fluorescence quantification of the tail of 54-hpf stat3^+/+, stat3^+/− and stat3^−/− larvae in Tg(gata1:dsRed)^sd2 transgenic background incubated in normoxia and hypoxia. Scale bar: 1 mm. n = 18 stat3^+/+ normoxia; 14 stat3^+/+ hypoxia; 41 stat3^+/− normoxia; 44 stat3^+/− hypoxia; 17 stat3^−/− normoxia; 20 stat3^−/− hypoxia (larvae used for this experiment came from four independent breeding between stat3^+/− zebrafish). E, F Representative pictures of 54-hpf control (ctrl inj) and hif1α morphants (MO-hif1α) in Tg(LysC:dsRed)^nz50 transgenic background incubated in normoxia and hypoxia for 6 h (scale bar: 500 μm) quantification of the ratio of dsRed-positive cells in region A and in region B. Area B between dashed lines. Arrowheads point at fluorescent cells in Area A. n = 8 ctrl inj normoxia; 9 ctrl inj hypoxia; 7 MO-hif1α normoxia; 8 MO-hif1α hypoxia (larvae used for this experiment came from three independent breeding between wild type zebrafish) G, H Representative pictures of 54-hpf stat3^+/+, stat3^+/− and stat3^−/− larvae in Tg(LysC:dsRed)^nz50 transgenic background incubated in normoxia and hypoxia for 6 h (scale bar: 500 μm); quantification of the ratio of dsRed-positive cells in region A and in region B. Area B between dashed lines. Arrowheads point at fluorescent cells in Area A. n = 11 stat3^+/+ normoxia; 16 stat3^+/+ hypoxia; 22 stat3^+/− normoxia; 23 stat3^+/− hypoxia; 11 stat3^−/− normoxia; 17 stat3^−/− hypoxia (larvae used for this experiment came from three independent breeding between stat3^+/− zebrafish). Mean ± SEM.