Fig 2: CA9 expression and activation of HIF1a signaling under acute and chronic hypoxia in MCF-7 and HCC1143 cell lines. A CA9 RNA expression in four datasets of transcriptome data, including MCF-7 24 h (MCF-7 24 h hypoxia normalized to MCF-7 24 h normoxia), HCC1143 24 h (HCC1143 24 h hypoxia normalized to HCC1143 24 h normoxia), MCF-7 5 days (MCF-7 5 days hypoxia normalized to MCF-7 5 days normoxia), HCC1143 5 days (HCC1143 5 days hypoxia normalized to HCC1143 5 days normoxia). Three biological replicates for each dataset; ****padj < 0.0001. B, C CA9 protein expression detected by western blotting (B) and Immunofluorescence of HIF1a localization by confocal microscopy (C) after 24 h (acute) and 5 days (chronic) incubation under normoxia (NX) and hypoxia (HX) in MCF-7 and HCC1143 cell lines. Blue, Hoechst; Green, HIF1a Ab. One representative experiment of three biological replicates is shown

Fig 3: Characterization of an antibody against human CA-IX. Figures show the binding of the antibody with CA-IX-positive sEVs (from HEK293pRTS-CA9 cells) (A) and with the recombinant protein (B).

Fig 4: Chronic hypoxia affects metabolism in luminal breast cancer cells. A, B Heatmap of genes involved in glycolysis (A) and cytoskeleton (B) pathways in 24 h (acute) and 5 days (chronic) hypoxia in MCF-7 and HCC1143 cells. NA, not available; ****padj < 0.0001; ***padj < 0.001; **padj < 0.01; *padj < 0.05; ns, not significant. White color means that genes were filtered out in targeted RNA sequencing data after filtering by DESeq2 package in R software. C Lactate levels (mM) in 6 cell lines under normoxia normalized to the OD value determined in SRB assay. D, E Lactate levels measured in three luminal (MCF-7, T47D, BT474) (D) and 3 basal A (HCC1143, SUM149PT, HCC1806) (E) breast cancer cell lines under acute normoxia/ hypoxia and chronic normoxia/ hypoxia normalized to the OD value determined in SRB assay. F CA9 RNA expression level under hypoxia in luminal and basal A cell lines detected by qRT-PCR. Log2(2^(-??CT)) was calculated by normalizing to normoxia in each cell line. Error bars indicate SD for triplicate measurements. ***p < 0.001; **p < 0.01; *p < 0.05. G GAPDH and PLOD2 protein expression detected by Western blot. B-actin serves as loading control. H Quantification of GADPH signal normalized to B-actin with Image J. Error bars indicate SD for triplicate measurements. **p < 0.01; *p < 0.05; ns, not significant

Fig 5: The CA-IX protein is associated with the membrane of hypoxic sEVs. EV identity was confirmed for all samples with the CD9 ELISA assay (A). Only A375 cells released CAIX-positive sEVs under hypoxia, and the CAI-X protein was associated with the EV membrane (B).