Extended Data Fig. 3: caMEK1 and caMKK6 induced senescence in vivo.
From: Characterizing primary and secondary senescence in vivo
a, Left: Immunostaining for NFκB in the caMEK1 and caMKK6 liver after 7 days of Dox treatment. Scale bars, 100 μm. Right: Quantification of NFκB-positive cells (n = 3 per group). b, Left: Immunostaining for NFκB in the caMEK1 and caMKK6 colon after 7 days of Dox treatment. Scale bars, 100 μm. Right: Quantification of NFκB-positive ratio in caMEK1 and caMKK6 after 3 and 7 days of Dox treatment (n = 3 per group, except caMKK6 day3 (n = 2)). c, qRT‒PCR analyses of the expression of senescence markers in the liver and colon of caMEK1 and caMKK6 chimeric mice at 3 and 7 days after Dox treatment (caMEK1 No Dox, n = 6; caMEK1 Dox 3 d, n = 7; caMEK1 Dox 7 d, n = 7; caMKK6 No Dox, n = 7; caMKK6 Dox 3 d, n = 6; caMKK6 Dox 7 d, n = 8). Expression levels relative to those in the liver or colon in the absence of Dox treatment are shown. Values that deviated more than twice the standard deviation from the mean were considered outliers and excluded. d, Schematic illustration of the genetic constructs for HDTVI; caMEK1 and HDTVI; caMKK6 for tissue-specific expression in the liver (upper panel). The lower panel shows the experimental protocol. e, RNA in situ analysis of Cdkn1a and mCherry gene expression in the HDTVI; caMEK1 and caMKK6 liver. Scale bars, 50 μm. n = 3 per group of mice examined. f, Quantification of the SA-β-gal index in HDTVI; caMEK1 and HDTVI; caMKK6 liver (HDTVI; caMEK1 No Dox, n = 5; Dox 7 d, n = 6; HDTVI; caMKK6 No Dox, n = 4; Dox, n = 6). g, Schematic illustration of the genetic constructs Villin-Cre; caMEK1 and Villin-Cre; caMKK6 for tissue-specific expression in the colon (upper panel). The lower panel shows the experimental protocol. h, Immunostaining for p21 and BrdU in Villin-Cre; caMEK1 and Villin-Cre; caMKK6 colon serial sections after 7 days of Dox treatment. Scale bars, 100 μm. i, Quantification of the p21- and BrdU-positive cells in Villin-Cre; caMEK1 and Villin-Cre; caMKK6 colons at 3 and 7 days after Dox treatment (Villin-Cre; caMEK1 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4; Villin-Cre; caMKK6 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4). j, qRT‒PCR analyses of the expression of Cdkn2a and Cdkn1a in Villin-Cre; caMEK1 and Villin-Cre; caMKK6 colons at 3 and 7 days after Dox treatment (Villin-Cre; caMEK1 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4; Villin-Cre; caMKK6 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4). Expression levels relative to those in the colon in the 3 days Dox treatment for Cdkn2a or the absence of Dox treatment for Cdkn1a are shown. Data are presented as the means ± s.d. of biologically independent samples. Unpaired t test, two sided (a, f). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, two sided (b, c, i, j).
