Fig 2: Drug resistant KG-1a cell line exhibits downregulation of miR-223 and upregulation of PKCε. (A) Cell viability of KG-1 and KG-1a cell lines after DOX treatment for 48 h, as detected by Cell Counting Kit-8 assay. (B) Relative miR-223 expression in KG-1 and KG-1a cells, as detected by RT-qPCR. (C) Relative PKCε protein level in KG-1 and KG-1a cells, as detected by western blot analysis. (D) Relative PRKCE mRNA expression in KG-1 and KG-1a cells, as detected by RT-qPCR. The values of KG-1 group in (B), (C) and (D) were normalized to 1 or 100% without accounting for standard error, due to the use of different batches for each replicate. **P<0.01; ***P<0.001 (n=3 replicates/group). The data were analyzed using unpaired Student's t-test. DOX, doxorubicin; miR, microRNA; PKCε/PRKCE, protein kinase C ε; RT-qPCR, reverse transcription-quantitative PCR.

Fig 3: Relative protein concentrations and normalized kinase statistic (s) of selected candidates of the Tyr (FGFR2, ZAP70) and Serine/Threonine (PKCepsilon, p38δ) kinase activity profiling. All blots were performed on a single membrane. After protein transfer (Fig. S1A), the membrane was cut at 55 kDa to obtain an upper section (>55 kDa) and a lower section (<55 kDa) (Fig. S1B). Both parts were then incubated with primary antibodies for the respective protein of choice according to their molecular weight in a sequential arrangement. A stripping step was performed after each membrane development step. Corresponding full-length blots and additional information are included in the supplementary information file (Figs. S1–S6). The upper section of the membrane was incubated in the following order: 1. PKCε (82 kDa) (Fig. S2); 2. ZAP70 (70 kDa) (Fig. S3); 3. FGFR2 (110 kDa) (Fig. S4); 4. Vinculin (130 kDa) (Fig. S5). The lower section was incubated with p38δ (Fig. S6). (a) Protein expression (left y-axis) and normalized kinase statistic (s) (right y-axis) of selected tyrosine kinases. The bars, shown in the top (left y-axis), represent the data from the densitometry analysis (mean ± SD) (n = 7/group). The protein levels of FGFR2 and ZAP70 were normalized to the corresponding Vinculin control and expressed relative to the protein level of the untrained group. Representative western blot analysis revealed no significant differences between the experimental groups (ET vs. UT). The blots of FGFR2 and ZAP70 are each grouped with vinculin which was measured in a different region of the same membrane using a different exposure condition in terms of antibody dilution, time and substrate for signal development (for details see supplementary information). Additionally, the results of normalized kinase statistic (s) were added on the right-axis for FGFR2 and ZAP70. The activity of both kinases was increased in lymphocytes of endurance-trained athletes (Qsp > 1.3). (b) Protein expression (left y-axis) and normalized kinase statistic (s) (right y-axis) of selected serine/threonine kinases. The bars, shown in the top (left y- axis), represent the data from the densitometry analysis (mean ± SD) (n = 7/group). The protein levels of PKCε and p38δ were normalized to the corresponding Vinculin control and expressed relative to the protein level of the untrained group. The relative protein level of p38δ was increased in ET (p ≤ 0.05). No significant differences between the experimental groups were demonstrated for PKCε (ET vs. UT). The blots of PKCε and p38δ are each grouped with vinculin which was measured in a different region of the same membrane using a different exposure condition in terms of antibody dilution, time and substrate for signal development (for details see supplementary information). In addition, the results of normalized kinase statistic (s) were added on the right-axis for PKCε and p38delta. The activity of PKCε was decreased in ET-derived lymphocytes (Qsp > 1.3). The activity of p38δ was slightly but not significantly increased in lymphocytes of endurance-trained athletes. PKCε: PKCepsilon; p38δ: p38delta; ET: endurance trained athletes, UT: untrained subjects.

Fig 4: Overexpression of miR-223 and inhibition of PKCε are not associated with the drug sensitivity of KG-1a cells. (A) Cell viability of transfected KG-1a cells after DOX treatment for 48 h, as detected by CCK-8 assay. (B) Relative PRKCE mRNA expression in KG-1a cells after transfection with PRKCE siRNAs, as detected by reverse transcription-quantitative PCR. (C) Relative PKCε protein level in KG-1a cells after transfection with si-PRKCE#3, as detected by western blot analysis. (D) Cell viability of PKCε-knocked down KG-1a cells after DNR treatment for 48 h, as detected by CCK-8 assay. The values of si-NC group in (B) and (C) were normalized to 1 or 100% without considering standard error, as each replicate was conducted using different batches. *P<0.05 vs. si-NC (n=3 replicates/group). The data were analyzed using unpaired Student's t-test or one-way ANOVA followed by Dunnett's post hoc test. DOX, doxorubicin; miR, microRNA; NC, negative control; si, siRNA; PKCε/PRKCE; protein kinase C epsilon; DNR, daunorubicin; CCK-8, Cell Counting Kit-8; NS, non-significant.

Fig 5: miR-223 targets and inhibits the expression of PKCε. (A) Relative miR-223 expression in KG-1a cells after transfection with miR-223 mimic, detected by RT-qPCR; mimic-NC values were normalized to 1. (B) Relative PRKCE mRNA expression in KG-1a cells after transfection with miR-223 mimic, as detected by RT-qPCR. (C) Relative PKCε protein level in KG-1a cells after transfection with miR-223 mimic, as detected by western blot analysis. (D) Binding sites between miR-223 and PRKCE mRNA predicted by TargetScan, and relative PKCε luciferase activity measured by dual-luciferase assay. The values of mimic-NC group in (B) and (C) were normalized to 1 or 100% without considering standard error, as each replicate was conducted using different batches. *P<0.05; **P<0.01 (n=3 replicates/group). The data were analyzed using unpaired Student's t-test. miR, microRNA; NC, negative control; PKCε/PRKCE, protein kinase C ε; WT, wild-type; Mut, mutant; UTR, untranslated region; RT-qPCR, reverse transcription-quantitative PCR.