Fig 2: Effect of MRP3 silencing in GSCs under normoxia and hypoxia. (A) MRP3 knockdown was confirmed by RT-qPCR (A) and Western blot (B). Viability of GSCs-MRP3KD was measured by MTS assay after a 24 h treatment with 0.5 µM doxorubicin (Dox) (C) or 0.5 µM teniposide (Tn) (D). n = 3, ** p < 0.01, *** p < 0.001, ### p < 0.001 relative to hypoxia control.

Fig 3: MRP3 expression in GSCs under hypoxic conditions. (A) MRP3 protein levels from GSCs under normoxia and hypoxia were analyzed by Western blot of GSCs. Bands were quantified and plotted. β-actin was used as a housekeeping control. (B) MRP3 expression was detected in human GBM samples by immunohistochemistry (scale bar 50 m). (C) Histograms represent fluorescence of CFDA accumulation (left) and plotted as fold of change of fluorescence (right). n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001, # p < 0.05 relative to hypoxia control.

Fig 4: Differential expression of genes associated with chemoresistance in GSCs depends on microenvironmental oxygen levels. (A) Chemoresistance in GSCs under normoxic conditions is elicited by the expression of the A3 adenosine receptor and MRP1 transporter (DOI: https://doi.org/10.3390/10.18632/oncotarget.12033, accessed on 26 May 2022). (B) In GSCs under a hypoxic microenvironment, A2B adenosine receptor expression increases. Activation of A2B triggers the expression of MRP3. Expression and activation of A2B potentially play a role in a novel mechanism of chemoresistance together with MRP3, which could be involved in GSC survival under hypoxia.

Fig 5: Role of adenosine in MRP3 expression in GSCs. GSCs were treated with 10 uM MRS1220 or 50 nM MRS1754. (A) A3 adenosine receptor protein levels in GSCs under normoxia and hypoxia were analyzed by Western blot. The bands were quantified and plotted. (B) As described in A, except for the A2B adenosine receptor. (C) MRP3 mRNA levels were analyzed by RT-qPCR. (D) A2B knockdown was confirmed by RT-qPCR. (E) MRP3 mRNA levels were analyzed by RT-qPCR in A2B knockout hypoxic GSCs. (F) MRP3 protein levels in GSCs were analyzed by Western blot. (G) Extrusion capacity was analyzed by flow cytometry using GSCs. Histograms represent the fluorescence of CFDA accumulation (left) and are plotted as a fold of change in fluorescence (right). n = 3, * p < 0.05, ** p < 0.01.