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. 2019 Dec 11;7:337. doi: 10.3389/fcell.2019.00337

FIGURE 4.

FIGURE 4

Rps15 is important for ribosome biogenesis and overexpression of Rps15 ameliorates senescent phenotypes in MEFs. (A) Relative quantification of Rps15 mRNA after knockdown Rps15 in MEFs. β-actin was used as internal control (Mean ± SEM, n = 3, ∗∗p < 0.01). (B) Polysomal profiles of shRps15 MEFs and negative control with continuous sucrose gradient of 10–50% were fractioned and measured with absorbance of light at 260 nm. Peaks belonged to large subunit of 60S, intact ribosome of 80S and polysomes were labeled. (C) Representative western blot analysis of untransfected control MEFs (CT) and overexpression of GFP, Rps15, Rps14, and Rps24 in extracts from respective stable-expression MEFs, recognized by anti-HA (mouse). β-actin was used as internal loading control. (D) Growth curves of Rps15, Rps24, Rps14, GFP stable transgenic MEFs and untransfected control MEF (CT) starting from passage 1 (Mean ± SEM, n = 3, ∗p < 0.05, ∗∗p < 0.01). (E) SA-β-gal positive rate of senescent Rps15, Rps24, Rps14, GFP stable-expression MEFs and untransfected control MEFs (CT) (Mean ± SEM, n = 3, ∗∗∗p < 0.001). (F) Relative quantification of p16INK4a mRNAs in senescent Rps15, Rps24, Rps14, GFP stable-expression MEFs and untransfected control MEFs (CT). β-actin was used as internal control (Mean ± SEM, n = 3). (G) A schematic model of mTORC1-Rps15 axis contributes in senescence-associated translation reduction.