Fig 1: Time-dependent expression of GSC markers following hypoxia(A) Real-time quantitative PCR indicated time-dependent changes of stem cell markers before (con) and after hypoxia in U87 glioma cells. In general, 6 h after hypoxia, there was a significant increase of stem cell markers, which reached peak values at 9–12 h. (*P < 0.05, One-sample T Test). (B) Western blot analysis indicated a higher expression of stem cell markers after hypoxia for 12–48 h in U87 glioma cells. (C) Gray value analysis of Western blot in B by Quantity One indicated the expression of stem cell markers (SOX-2, OCT-4, KLF-4, Nanog, CD133, CD15, NESTIN and ABCG2) increased at least two-fold compared with control (*P < 0.05, One-sample T Test). (D) An increase expression of CD133, CD15 and NESTIN with a time-dependent manner after hypoxia (*P < 0.05, One-sample T Test).
Fig 2: Hypoxia-induced neurospheres exhibited high expression of stem cell markers via immunofluorescence staining(A) Neurospheres formed by single CD133-CD15-NESTIN- GL261 cell under hypoxia exhibited high expression of stem cell markers (SOX-2, OCT-4, KLF-4, Nanog, CD133, CD15, NESTIN and ABCG2). (B) The expression of stem cell markers of GBM CD133-CD15-NESTIN- glioma cells exposed in hypoxia (1% O2) 48 h was higher at least 1.5-fold compared with normoxia (21% O2) (*P < 0.05, Paired-samples T Test).
Fig 3: The expression of FBI-1 or PXR in patient-derived TNBC cells with mutated BRCA.A Ten lines of TNBC patient-derived cells (TNBC No. 1 to No. 10) were cultured and harvested for western blot experiments or qPCR. The endogenous protein levels of FBI-1 or PXR were examined by their antibodies and the results were shown as images of Western blot, quantitative analysis of images (mean ± SD) or the mRNA level of FBI-1 or PXR (mean ± SD). B FBI-1 enhanced the expression of protein levels in PXR’s downstream multi-drug resistance-related genes, BCRP and MDR-1, in patient-derived TNBC cells. TNBC PDC No. 3 was transfected with FBI-1 vector or the siRNA of FBI-1. The cells were cultured by using phenol red-free DMEM and activated carbon-treated serum (0.5%) and treated with 10µmol/L concentration of rifampicin. The protein samples were extracted and analyzed by western blot tests. The protein levels of FBI-1, BCRP, or P-GP were identified by their antibodies. The GAPDH was chosen as the loading control.
Fig 4: The association between FBI-1 and factors related to the PXR pathway.The mRNA level of PXR, BCRP, MDR-1, miR-30c, or FBI-1 in TNBC clinical specimens was examined using the qPCR experiments. The association between the expression of FBI-1 with PXR, BCRP, miR-30c, or MDR-1 was examined. The results were shown as scatter-plot images. The abscissa is the expression level of FBI-1; the ordinate is the corresponding expression level of PXR, BCRP, MDR-1, or miR-30c.
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