Fig. 4

A Fnip2^C Knock-in mouse to model FNIP2 rs2291007. A Experimental strategy to generate a knock-in mouse model mimicking FNIP2 rs2291007C using CRISPR Cas9 gene-editing tool in blastocysts. B RT-qPCR from liver and gonadal white adipose tissue (WAT) obtained from Fnip2^T/T (n=8) and Fnip2^C/C (n=9) mice ad libitum fed. Fnip2 mRNA levels are normalized to β-actin levels and relativized to the levels in Fnip2^T/T. Statistical significance was calculated by using unpaired two-tailed t-test. C Fnip2 mRNA levels were measured by RT-qPCR in mouse embryonic fibroblasts (MEFs) derived from Fnip2^T/T (n=5) and Fnip2^C/C (n=5) mice in complete Roswell Park Memorial Institute medium (RPMI; supplemented with dialyzed serum) or RPMI without amino acids for 2 h. Fnip2 mRNA levels are normalized to β-actin levels and relativized to the levels in Fnip2^T/T +AA. D MEFs derived from Fnip2^T/T and Fnip2^C/C mice were deprived of all amino acids (AA) in RPMI supplemented with dialyzed serum for 45 min and re-stimulated with AA for 5, 10, and 20 min. Whole-cell protein lysates were immunoblotted for the indicated proteins. E MEFs derived from Fnip2^T/T (n=5) and Fnip2^C/C (n=5) mice were treated with 5μg/mL Actinomycin D for 2 and 4h. Fnip2 mRNA levels were measured by RT-qPCR, normalized to β-actin levels, and then made relative to Fnip2 levels in untreated samples. Statistical significance was calculated using 2-way ANOVA with Sidak’s multiple comparison correction. F The area under the curve (AUC) was calculated from E and statistical significance was calculated by using unpaired two-tailed t-test