Extended Data Fig. 1. Rragc-mutant mice have shortened lifespan.
a. Chromatogram of the Rragcwt and RragcS74N allele. RragcS74N allele encodes two amino acid substitutions (blue bold), plus silent diagnostic mutations (purple). b. Progenies of RragcS74N breeding schemes. c. MEFs from Rragc+/+ and RragcS74N/+ mice were deprived of all amino acids in RPMI medium with dialyzed serum for the indicated time-points. d. Quantification of P-T389-S6K1 and TFEB upper band (phosphorylated) from triplicates from Fig. 1a and Figure S1c (Statistical significance was calculated by two-tailed Student’s t-test for AUC). Data are presented as mean values ± SD. e. (Left) MEFs from Rragc+/+ (n = 4 biologically independent cells), RragcS74N/+ (n = 4 biologically independent cells), and RragcS74N/S74N (n = 2 biologically independent cells), mice were deprived from and re-stimulated with amino acids for 10 min. (Right) Quantification of P-T389-S6K1 and TFEB upper band in -AA samples. Data are presented as mean values ± SD f. (Left) Immunofluorescence of TFEB in Rragc+/+ and RragcS74N/S74N MEFs in RPMI with and without amino acids; (Right) with the percentage of nuclear TFEB per cell (Rragc+/+ + AA n = 112 –AA n = 117; RragcS74N/S74N + AA n = 72 –AA n = 91). Bars represent 100μm g. TFE3 and TFEB levels fractions of hearts from Rragc+/+ (n = 3) and RragcS74N/+ (n = 3) mice after fasting. h. Quantification of TFE3 and TFEB levels in the nucleus. Data are presented as mean values ± SD. i. Representative anti-TFEB IHC (left) and quantification (right) of TFEB-positive cells in the cytoplasm (Ct), nucleus (N) and cytoplasm&nucleus (Ct&N) in renal tubules from young Rragc+/+ (n = 4) and RragcS74N/+ (n = 4) male mice. Scale bar 100 µm. j. Kaplan–Meier survival curves of Rragc+/+ (n = 18) and RragcS74N/+ (n = 20) male mice (left), Rragc+/+ (n = 19) and RragcS74C/+ (n = 13) male mice (center) and Rragc+/+ (n = 18) and RragcT89N/+ (n = 19) male mice (right). k. Kaplan–Meier survival curves of Rragc+/+ (n = 21) and RragcS74N/+ (n = 24) female mice (left), Rragc+/+ (n = 19) and RragcS74C/+ (n = 23) female mice (center) and Rragc+/+ (n = 17) and RragcT89N/+ (n = 22) female mice (right). l. Kaplan–Meier survival curves of Rragc+/+ (n = 9) and RragcS74N/+ (n = 24) mice that were free of detectable malignant tumors at the time of death. m. Kaplan–Meier survival curves of Rragc+/+ (n = 10) and Rragc S74C/+ (n = 17) mice without malignant tumors. In j-m, statistical significance was calculated with the log-rank test. n. Bone mineral density (BMD) measured in 13.5-21.5-month-old Rragc+/+ (n = 17) and RragcS74N/+ (n = 21) male mice. o. Blood pressure in 16-19-month-old Rragc+/+ (n = 6) and RragcS74N/+ (n = 7) male mice. p. Hematological parameters in 3–5- and 18-month-old Rragc+/+ (young, n = 8; old, n = 16) and RragcS74N/+ (young, n = 9; old, n = 14) male mice. q. Representative anti-p21 IHC in kidneys from 18-month-old Rragc+/+ and RragcS74N/+ male mice. Bars represents 20 μm. Arrows indicate p21-positive cells. r. Representative anti-p21 IHC (left) and quantification (right) from pancreas harvested from 18-month-old Rragc+/+ (n = 3) and RragcS74N/+ (n = 5) male mice. Bars represent 200 μm. Statistical significance in e, h, n, p and r was calculated by two-tailed Student’s t-test. Statistical significance in f was calculated by one-way ANOVA and Tukey’s multiple comparisons test. Statistical significance in a and i was calculated by by two-sided Fisher’s exact test. Statistical significance in o was calculated by Two-way Anova.