Fig 1: Effect of candidate variants NRAS p.(Gln61Lys), PTPN11 p.(Tyr279Cys) and PDGFRB p.(Glu578Gln) on the response to TKI treatment. (A) Graphical representation of the pathways affected by variants in the candidate genes NRAS/KRAS, PTPN11 (encoding SHP2), PDGFRB and KMT2D. (B–D) Top left: Western Blot of successful transfection of wild-type (WT) and variant into TKI-sensitive K-562 cells compared to GAPDH. Cellular fitness after WT and variant transfection and 48 h nilotinib exposure (0.1 µM) for (B) NRAS WT and p.(Gln61Lys), as well as imatinib exposure (2 µM) for (C) PTPN11 WT and p.(Tyr279Cys) and (D) PDGFRB WT and p.(Glu578Gln). Top right: Total cell number analyzed using trypan blue staining. Bottom left: Metabolic activity measured by WST assay. Bottom middle: Caspase 9 activity analyzed by caspase 9-Glo assay. Bottom right: Ki-67 expression to investigate cellular proliferation. Data was normalized to respective negative control (NC) and analyzed using Two-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: IL-15–induced PDGFRβ expression is mediated by PI3K/AKT signaling. (A and B) Primary NK cells were treated with IL-15 (10 ng/mL) for the indicated times. mRNA levels of PDGFRB at different time points (A) (n = 3) or at 1 h (B) (n = 10) were examined by qPCR. (C and D) Primary NK cells were pretreated with ActD (5 μg/mL) (C) or cycloheximide (CHX, 20 μg/mL) (D) for 1 h, washed twice with RPMI-1640, and then treated with IL-15 (10 ng/mL) for 24 h. Dimethyl sulfoxide (DMSO) was used as a control. Expression levels of PDGFRβ were examined by flow cytometry (n = 5). Data shown are representative histograms and percentage of inhibition with the following equation: % inhibition = 100 × [1 − (DMSO-inhibitor)/DMSO]. (E) Primary NK cells were pretreated with wortmannin (1 μM), afuresertib (10 μM), TPCA-1 (1 μM), rapamycin (10 μM), torin1 (10 μM), decernotinib (10 μM), C118-9 (10 μM), STAT5-IN-1 (10 μM), AZD6244 (10 μM), or CI-1040 (10 μM) for 1 h, washed twice with RPMI 1640, and then treated with IL-15 (10 ng/mL) for 24 h. DMSO was used as control. Data shown are percent of inhibition (n = 5). The mean value of the inhibitory rate is shown. (F) Luciferase reporter assay shows that p65 activates PDGFRB gene transcription. (G–I) Binding of p65 to the PDGFRB promoter in IL-15–treated NK cells (G) or resting NK cells (H) as determined by ChIP-qPCR or PCR (I) (n = 3). Data represent three independent experiments. Data shown are means ± SD. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 3: Influence of PTPN11 p.(Tyr279Cys) and PDGFRB p.(Glu578Gln) on the development of imatinib resistance. Stably transfected cells expressing either PTPN11 wild-type (WT), p.(Tyr279Cys), or PDGFRB WT or p.(Glu578Gln) were exposed to increasing concentrations of imatinib. (A) Cells were cultivated with the respective imatinib concentration and the total cell number was analyzed using trypan blue staining for 0.1, 0.2, and 0.3 µM imatinib within 21 days. Black: Negative control (NC); dark grey: PTPN11; light grey: PDGFRB; solid line: mutation; dashed line: WT. (B) Ki-67 expression to analyze proliferation and (C) Caspase 9 activity of PTPN11 WT and p.(Tyr279Cys) transfected cells after 21 days of treatment with the respective imatinib concentration measured by caspase 9-Glo assay. Data were normalized to NC. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. ***p < 0.001.
Supplier Page from Sino Biological, Inc. for Human PDGFRB/CD140b Gene ORF cDNA clone in cloning vector