Fig 2: PAL reduced MM tumors in the mouse mode. (A) PAL could significantly decreased the expression of DHFR in U266 mice (**P<0.01); (B) PAL significantly increased the expression level of apoptosis marker caspase3 in U266 mice (*P<0.05); (C) tumor morphology of U266 mice treated with PAL, unit of ruler is cm; (D) PAL remarkably decreased the tumor size in U266 mice (**P<0.01; ***P<0.001); (E) PAL could significantly decrease the expression of DHFR in KMS27 mice (*P<0.05); (F) PAL significantly increased the expression level of apoptosis marker caspase9 in KMS27 mice (*P<0.05); (G) tumor morphology of U266 mice treated with PAL; (H) PAL also remarkably decreased the tumor size in KMS27 mice (*P<0.05; **P<0.01). Unit of ruler is cm. PAL, palbociclib; MM, multiple myeloma; DHFR, dihydrofolate reductase.

Fig 3: PAL inhibits the growth of MM cells by targeting DHFR. (A) SiRNA against DHFR significantly knocked down the mRNA expression in the U266 cell line (*P<0.05); (B) siRNA against DHFR markedly reduced the protein level of DHFR in the U266 cell line; (C) siDHFR alone could increase the mRNA expression of MCM2 in the U266 cell line, while PAL did not further increase the expression level of MCM2 in the U266 cell line under DHFR knockdown (*P<0.05, NS indicated not significant); (D) siDHFR alone did not decrease the cell viability of the U266 cell line, while PAL could not further decrease the cell viability of the U266 cell line under DHFR knockdown (*P<0.05, NS indicated not significant); (E) siRNA against DHFR significantly knocked down the mRNA expression in the KMS27 cell line (*P<0.05); (F) siRNA against DHFR significantly reduced the protein level of DHFR in the KMS27 cell line; (G) siDHFR alone could increase the mRNA expression of MCM2 in the KMS27 cell line, while PAL did not further increase the expression level of MCM2 in the KMS27 cell line under DHFR knockdown (*P<0.05, NS indicated not significant); (H) siDHFR alone did not decrease the cell viability of the KMS27 cell line, while PAL could not further decrease the cell viability of the KMS27 cell line under DHFR knockdown (*P<0.05, NS indicated not significant). PAL, palbociclib; MM, multiple myeloma; DHFR, dihydrofolate reductase. “+” is with agents; “−” is without agents.

Fig 4: PAL significantly decreased the expression of DHFR in MM cell lines. (A) PAL decreased the mRNA expression of DHFR in U266 cells in a dose-dependent manner (*P<0.05); (B) PAL reduced the protein level of DHFR in U266 cells; (C) PAL decreased the mRNA expression of DHFR in KMS27 cells in a dose-dependent manner (*P<0.05); (D) PAL reduced the protein level of DHFR in KMS27 cells; (E) PTX increased the mRNA expression of DHFR in U266 cells in a dose-dependent manner (*P<0.05); (F) PTX increased the protein level of DHFR in U266 cells; (G) PTX increased the mRNA expression of DHFR in KMS27 cells in a dose-dependent manner (*P<0.05); (H) PTX increased the protein level of DHFR in KMS27 cells; (I) MTX increased the mRNA expression of DHFR in U266 cells in a dose-dependent manner (*P<0.05); (J) MTX increased the protein level of DHFR in U266 cells; (K) MTX increased the mRNA expression of DHFR in KMS27 cells in a dose-dependent manner (*P<0.05); (L) MTX increased the protein level of DHFR in KMS27 cells. PAL, palbociclib; DHFR, dihydrofolate reductase; PTX, pralatrexate; MM, multiple myeloma; MTX, methotrexate.

Fig 5: Impact of CSF on the expression of methotrexate target proteins in leukemia cells. (A) Immunoblots showing the effects of CSF on methotrexate target proteins. Leukemia cell lines were cultured in regular media or CSF for 48 hours. Protein lysates were then collected for immunoblotting with DHFR, TYMS, ATIC, or β-actin antibodies. (B-D) Dose-response curves for wild-type (WT) DHFR or mutant DHFR (mDHFR; L22F, F31S) NALM-6 (B), REH (C), and Jurkat (D) leukemia cells treated with different concentrations of the methotrexate. Leukemia cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. LogIC50 and bottom values with confidence intervals were calculated from the dose-response curves and are shown in the supplemental Table.