Fig 2: The expressions of BCR-ABL oncoprotein and its downstream signaling molecules were decreased in RFNs treated CML cells. K562 and K562/G01 cells were co-delivered with gRNA-17 plus donor, RFNs-half plus donor, RFNs plus donor, respectively. Proteins were extracted after 60 h of transfection and analyzed by western blot. Expressions of p-BCR-ABL and BCR-ABL were reduced in RFNs plus donor group (a). The activated phospho-CRKL, phospho-ERK and phospho-STAT5 were down-regulated in RFNs plus donor group (b)

Fig 3: Combination treatment of GNF5 and docetaxel induces cytoplasmic localization of Yap1 and decreases expression of downstream transcription targets of Yap1 compared to vehicle control treated lung adenocarcinomasGFP+ cells were isolated from KRASG12D/+; p53-/-; Rosa26-fGFP mice two weeks after treatment with vehicle, docetaxel, GNF5, or combination treatment and 12 weeks after induction of tumors with adenovirus. A. Immunofluorescence staining for Yap1 shows a decrease in Yap1 nuclear localization in mice treated with GNF5 and docetaxel compared to vehicle control treated mice. Scale = 75 µm. B. Higher magnification images are provided to show sub-cellular localization of Yap1 in control and combination treatment mice. Scale = 7.5 µm. C. Western blot analysis showed no significant difference in total Yap1 protein expression in double treated mice compared to vehicle treated control mice. Phospho-CrkL expression is a marker of ABL kinase activity. D. RT-qPCR analysis showed a decrease in mRNA transcript expression of the downstream Yap1 target, Birc5, which encodes the protein survivin, in mice treated with both GNF5 and docetaxel compared to vehicle control treated mice or mice treated with GNF5 or docetaxel alone (n = 3-4 mice per group, each RT-qPCR assay performed in triplicate). E. Western blot analysis showed a decrease in protein expression of downstream transcriptional targets of Yap1, including survivin, c-Myc, S100A4, and cyclin D1. Graphs depict means and S.E.M.

Fig 4: Effects of the lack of glutamine on glucose consumption, lactate production, pH reduction and BCR/Abl expression/signaling in low oxygen. K562 (A,C,E,G) or KCL22 (B,D,F,H) cells were seeded at 3 × 105 cells/mL and incubated in atmosphere at 0.2% O2 for the indicated times in the absence (0/-) or the presence (2/+) of 2 mM glutamine from time 0 (t0) of incubation. (A–F) Glucose and lactate concentrations and pH were measured in culture medium. Values are mean ± S.D. of data obtained from 3 independent experiments; 0 vs. 2 mM glutamine: * p < 0.05, ** p < 0.01. Horizontal bars indicate the t0 values. (G,H) Total cell lysates in Laemmli buffer were obtained at the indicated times of incubation and subjected to SDS-PAGE and immuno-blotting with anti-Abl or anti-p-Crkl Ab; anti-ARD1 Ab was used to verify the equalization of protein loading. One representative experiment out of 3 with similar results is shown. The original Western blotting images and the relative densitometry values can be found in Supplementary Figure S1.

Fig 5: Effects of 3PO or 6-AN on BCR/Abl signaling and maintenance of stem cell potential in low oxygen in the absence or the presence of glutamine. K562 (A,C,E) or KCL22 (B,D,F) cells were seeded at 3 × 105 cells/mL and incubated in atmosphere at 0.2% O2 for 4 or 7 days, respectively, in the absence (0) or the presence (2) of 2 mM glutamine and with or without (-/+) the indicated compounds, added at time 0 of incubation (LC1). (A,B) Total cell lysates in Laemmli buffer were subjected to SDS-PAGE and immuno-blotting with anti-p-Crkl Ab or anti-vinculin Ab, used to verify equalization of protein loading. One representative experiment out of 3 with similar results is shown. (C–F) LC1 cells were replated at 3 × 104 cells/mL into drug-free LC2 incubated at 21% O2 and in any case in the presence of glutamine. Trypan blue-negative cells were counted at the times of incubation in LC2 indicated in abscissa. Values are mean ± S.D. of data obtained from 3 independent experiments; * p < 0.05, ** p < 0.01 vs. 2 mM glutamine/no drug. The original Western blotting images and the relative densitometry values can be found in Supplementary Figure S3.