Fig 2: MKK3 and MKK6 kinases in ERMS cells. Summary diagram describing the differential role played by MKK3 and MKK6 in controlling growth arrest and myogenic differentiation in ERMS cells. MHC, myosin heavy chain.

Fig 3: MKK3 and MKK6 gene expression levels in ERMS and NSM samples. Using the R2-Genomics Analysis and Visualization Platform, MKK3 or MKK6 gene expression was assessed in ERMS primary tumours or NSM across different datasets: (A) Barr and Assmann datasets, (B) Davicioni and Hofman datasets, (C) Schafer Welle dataset. Statistical analysis was performed using one-way ANOVA. (A-C) Right panels illustrate the relative expression of MKK3 and MKK6 in ERMS samples. ERMS, embryonal rhabdomyosarcoma; NSM, normal skeletal muscle.

Fig 4: The effects of MEK6-AS1 on the adipogenic differentiation of hAMSCs. a MEK6-AS1 was silenced in hAMSCs using two independent pair of siRNAs (si-MEK6-AS1-1 and si-MEK6-AS1-2). The knockdown efficiency was verified by qRT-PCR as compared with the negative control (si-NC). b The expression of adipogenic markers in hAMSCs with MEK6-AS1 knockdown and in control hAMSCs on day 6 after adipogenic induction were analyzed by qRT-PCR. c Western blot analysis was performed to validate the expression of adipogenic markers in MEK6-AS1 knockdown hAMSCs or control hAMSCs on days 6 after adipogenic induction. d and e Oil Red O staining was used to confirmed the generation of lipids in cells treated with MEK6-AS1 siRNAs and corresponding control cells on day 10 after adipogenic induction (d) and the quantification of Oil Red O staining (e). Scale bars: 100 µm. f and g hAMSCs were transduced with lentivirus overexpressing MEK6-AS1 (lenti-MEK6-AS1) or control lentivirus (lenti-NC), and overexpression efficiency was detected by fluorescence observation (f) and qRT-PCR (g). Scale bars: 200 µm. (h and i) qRT-PCR (h) and western blot (i) analysis were performed to validate the expression of adipogenic markers in MEK6-AS1-overexpressed cells and corresponding control cells on day 6 after adipogenic induction. j and k The gerenation of lipids in cells on day 10 after adipogenic induction were indicated by Oil Red O staining (j) and quantification of the Oil Red O staining (k). Scale bars: 100 µm. GAPDH was used as internal control for qRT-PCR and western blot. The quantitative data were normalized to GAPDH, n = 3. Data are shown as the mean ± SD. Statistically significant differences were considered as follows: ∗P < 0.05, ∗ ∗P < 0.01, ∗ ∗ ∗P < 0.001, ∗ ∗ ∗ ∗P < 0.0001

Fig 5: Morphological and functional changes induced by MKK6 or MKK3 overexpression in RD cells. (A) Western blots showing MKK3 and MKK6 expression as the control of the transfection with the specific vectors. Tubulin was used as a loading control. The numbers on the left of the blots indicate the protein size (kDa). (B) Differences in viable RD cell number in cells transfected with empty vector (CMV), caMKK3 or caMKK6 assessed using the trypan blue exclusion assay. Histograms represent the mean value ± SD of three independent experiments. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test: **P<0.01; *P<0.05 vs. CMV. (C) Phase contrast images of RD cells transfected with CMV, caMKK3 or caMKK6. MKK6 induces typical elongated myogenic morphology not present in caMKK3 transfected RD cells. (D) CMV-, caMKK3- or caMKK6-transfected cells after immunofluorescence staining with Myc or MHC antibodies. DAPI was used for nuclear staining. Scale bars, 50 µm. Experiments were performed twice. MHC, myosin heavy chain.