Fig. 6: Mutation of miR-802 binding sites inTmed9 is sufficient to phenocopy the Vil-Cre mir-802^fl/fl Paneth cell phenotype.

a Fraction (%) of Tmed9 mutant allele expression on Tmed9 expression in the intestinal epithelium of Vil-Cre Tmed9KI^mut/mut, Vil-Cre Tmed9KI^wt/mut, and control Tmed9KI^mut/mut mice. Box plot defined by minima (0, 58.43, 95.46), median (1.377, 60.55, 96.21), maxima (2.5, 61.39, 98.98), 25% percentile (0,58.43, 95.6), 75% percentile (2.5, 61.39, 98.33), range (2.5, 2.957, 3.520), mean (1.292, 60.12, 96.71) for genotypes Tmed9KI^mut/mut,Vil-Cre Tmed9KI^wt/mut, Vil-Cre Tmed9KI^mut/mut respectively with line shown at mean (n = 3, 3, 4 respectively per genotype). b Relative mRNA expression, measured by qRT-PCR, of Tmed9 in the isolated intestinal epithelium of Vil-Cre Tmed9KI^mut/mut, Vil-Cre Tmed9KI^wt/mut, and control Tmed9^mut/mut mice (n = 3,3,4 for each genotype respectively). Data are representative of three independent experiments. c Immunoblot analysis of TMED9 in the jejunum of Vil-Cre Tmed9KI^mut/mut Vil-Cre Tmed9KI^wt/mut, and control Tmed9KI^mut/mut mice. β-ACTIN was used as a loading control. Quantitative densitometric analysis is shown on the right. Each lane represents a lysate from a different mouse (n = 3 per genotype). Data are representative of two independent experiments. d Oral glucose tolerance test of Vil-Cre Tmed9KI^mut/mut and control Tmed9KI^mut/mut mice (n = 7 for each group, two independent experiments). e Relative mRNA expression levels, measured by qRT-PCR, of antimicrobial peptides in jejunum of Vil-Cre Tmed9KI^mut/mut and Vil-Cre mir-802^fl/fl compared to control mice (Tmed9KI^mut/mut and mir-802^fl/fl) (n = 13, 6, 5 for Tmed9KI^mut/mut and mir-802 ^fl/fl, Vil-Cre Tmed9KI^mut/mut, and for Vil-Cre mir-802 ^fl/fl, respectively for each group). Data are representative of two independent experiments. f Immunoblot analysis of LYZ in isolated enterocytes from upper jejunum of Vil-Cre Tmed9KI^mut/mut and Tmed9KI^mut/mut littermates. The quantification of densitometric analysis is shown on the right. Each lane represents a lysate from a different mouse (n = 4 per genotype, two independent experiments). g Tmed9 transcript levels, measured by qRT-PCR, in FACS-sorted Paneth cells of Vil-Cre Tmed9KI^mut/mut mice compared to Tmed9KI^mut/mut controls (n = 4 per genotype, one experiment). h, i Immunoblot analysis of LYZ in C2BBe1 cells that were infected with Ad-Tmed9 (h) or transfected with siRNAs targeting Tmed9 or control siRNAs (i). β-ACTIN was used as a loading control. Quantitative densitometric analysis is shown on the right (n = 3 per group, one experiment). j Immunoblot analysis of LYZ in C2BBe1 cells infected with Ad-miR-802 and control (Ad-GFP) adenovirus. β-ACTIN was used as a loading control. Quantitative densitometric analysis is shown on the right (n = 3 per group). Data are representative of two independent experiments. Data are plotted as mean ± SD. Statistical significance was evaluated by descriptive statistics (a) unpaired two-tailed t-tests (f–j), by one-way ANOVA followed by Tukey’s multiple comparison test (b, c, e), or two-way ANOVA for repeated measures with Sidak’s multiple comparisons test (d).