Fig. 4

Reversal of MDR ability via knockout of target P110 subunits, including P110α (PIK3CA) and P110β (PIK3CB), from MDR cancer cell populations of KB-C2 and H460/MX80 with pCRISPR-CG01 all-in-one plasmid. a Map of pCRISPR-CG01. b Analysis with Western blot confirming the knockout of PIK3CA and PIK3CB that is of low abundance in KB-C2 and H460/MX80, as compared with GAPDH. To correct possible result deviation caused by exposure saturation of partial bands, less exposure of GAPDH bands was used to indicate relative cell counts. The absence or intensity-reduction of the target bands are depicted by red stars. Truncated proteins are depicted by head-down arrows. Relative quantification was carried out with ImageQuant TL based on the intensity of the bands. Statistical calculation was made based on three independent repeats. c PCR as adjuvant method to characterize target P110 subunit deficiency in KB-C2 and H460/MX80 cells. Missing bands or reduced copies of the target PCR products are depicted by red stars. New bands generated by chromosomal recombination are depicted by diamonds. Truncated fragments are depicted by head-down arrows. d-f MTT assay showing changes in MDR level of the KB-C2 and H460/MX80 cells with target PI3K 110α or 110β subunits knocked out. Colchicine and paclitaxel, the substrates of P-gp, were used for evaluation of reversal of KB-C2 over-expressing P-gp. The BCRP substrate mitoxantrone was used for analysis of reversal of MDR of H460/MX80 with BCRP over-expressed. The experiments were performed at least three times