Figure 6.
Overactivation of mTORC1 pathway acts as a compensatory mechanism for rRNA degradation. (A) Schematic overview of the mTORC1 pathway. We hypothesized that increased rRNA degradation mediated by SLFN14 K219N stimulates mTORC1 signaling as a compensatory mechanism. Active mTORC1 signaling stimulates ribosomal biogenesis via phosphorylation of S6K1-T389. In addition, mTORC1 is involved in cell growth, survival, motility, and mitochondrial biogenesis. Figure created using BioRender.com. (B) Representative immunoblots showing phosphorylated S6K1-T389 protein expression in undifferentiated cells and day 4 MK for WT/WT, WT/K219N, and K219N/K219N conditions (left). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control for normalization. Quantification of immunoblot data for 6 (day 0) and 7 (day 4) independent differentiation experiments show a significant increase in S6K1-T389 phosphorylation in WT/K219N and K219N/K219N day 4 MK compared with WT/WT, whereas no difference was seen in undifferentiated cells (day 0). Analysis performed on cells between passage weeks 10 to 13 for WT/WT and between weeks 3 and 6 for WT/K219N and K219N/K219N (right). Statistics were performed with a pairwise Wilcoxon test for all comparisons. (C) Immunoblot analysis showing a complete lack of S6K1-T389 phosphorylated protein in day 4 MK treated with rapamycin (and without) at passage week 5. GAPDH was used as a loading control. (D) Gel electrophoresis analysis shows rapamycin treatment exacerbates rRNA degradation in SLFN14 K219N cells. Analysis of cells at passage week 5. (E) Electrophoresis traces of rapamycin-treated cells show increased RNA degradation fragment peaks (red arrows) and reduced 28S peaks (light blue arrows) in WT/K219N cells and an even stronger effect in K219N/K219N day 4 MK. Analysis performed on cells at passage week 5. (F) Treatment with rapamycin significantly reduces the area of the 28S peak for both WT/K219N and K219N/K219N on day 4 MK. Analysis performed on cells between passage weeks 3 to 5. Statistics were performed with a pairwise Wilcoxon test for all comparisons. Boxplots for all panels represent the middle 50% of the data points, with 25% outliers as whiskers and the mean as a black horizontal line. All individual data points were added as dots to the graph. FU, fluorescence units; NT, nucleotides.

Overactivation of mTORC1 pathway acts as a compensatory mechanism for rRNA degradation. (A) Schematic overview of the mTORC1 pathway. We hypothesized that increased rRNA degradation mediated by SLFN14 K219N stimulates mTORC1 signaling as a compensatory mechanism. Active mTORC1 signaling stimulates ribosomal biogenesis via phosphorylation of S6K1-T389. In addition, mTORC1 is involved in cell growth, survival, motility, and mitochondrial biogenesis. Figure created using BioRender.com. (B) Representative immunoblots showing phosphorylated S6K1-T389 protein expression in undifferentiated cells and day 4 MK for WT/WT, WT/K219N, and K219N/K219N conditions (left). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control for normalization. Quantification of immunoblot data for 6 (day 0) and 7 (day 4) independent differentiation experiments show a significant increase in S6K1-T389 phosphorylation in WT/K219N and K219N/K219N day 4 MK compared with WT/WT, whereas no difference was seen in undifferentiated cells (day 0). Analysis performed on cells between passage weeks 10 to 13 for WT/WT and between weeks 3 and 6 for WT/K219N and K219N/K219N (right). Statistics were performed with a pairwise Wilcoxon test for all comparisons. (C) Immunoblot analysis showing a complete lack of S6K1-T389 phosphorylated protein in day 4 MK treated with rapamycin (and without) at passage week 5. GAPDH was used as a loading control. (D) Gel electrophoresis analysis shows rapamycin treatment exacerbates rRNA degradation in SLFN14 K219N cells. Analysis of cells at passage week 5. (E) Electrophoresis traces of rapamycin-treated cells show increased RNA degradation fragment peaks (red arrows) and reduced 28S peaks (light blue arrows) in WT/K219N cells and an even stronger effect in K219N/K219N day 4 MK. Analysis performed on cells at passage week 5. (F) Treatment with rapamycin significantly reduces the area of the 28S peak for both WT/K219N and K219N/K219N on day 4 MK. Analysis performed on cells between passage weeks 3 to 5. Statistics were performed with a pairwise Wilcoxon test for all comparisons. Boxplots for all panels represent the middle 50% of the data points, with 25% outliers as whiskers and the mean as a black horizontal line. All individual data points were added as dots to the graph. FU, fluorescence units; NT, nucleotides.

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