Pcr primers

Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA). PCR was performed using Herculase DNA polymerase (Stratagene). PCR primers were obtained from Integrated DNA Technologies. All the plasmids used in this study are derivatives of p2TK2-SW2 mCh(Gro) (Agaisse and Derre, 2014 (link)). They express mCherry under the control of the groESL operon promoter and terminator, the TetR repressor and the indicated Inc under the control of the tetA gene promoter and incDEFG operon terminator. The p2TK2-SW2 mCh(Gro) Tet IncD-, IncE-, and IncG-3xFLAG plasmids were described previously (Agaisse and Derre, 2014 (link); Mirrashidi et al., 2015 (link)). p2TK2-SW2 mCh(Gro) Tet IncD-Myc, CTL0314-3xFLAG, CTL0475-3xFLAG, or IncD/IncE-3xFLAG chimera were constructed similarly using the primers listed in Supplementary Table S1.

Chemicals and other reagents were obtained from Thermo Fisher Scientific and Sigma-Aldrich. Radioactive isotopes were acquired from PerkinElmer Life Sciences (Waltham, MA). The IDT 20/100 ladder, PCR primers, and fluorescent hydrolysis probes were obtained from Integrated DNA Technologies (Coralville, IA), and oligomers for transcription template preparation, plasmid modification, and vector synthesis were from Sigma-Aldrich. The 50-bp DNA ladder and enzymes other than those indicated below were obtained from New England Biolabs (Ipswich, MA).

All cloning DNA oligonucleotides and PCR primers were obtained from Integrated DNA Technologies (IDT) (see Supplementary Tables 1 and 2). Restriction enzymes were purchased from New England Biolabs (NEB) and used according to the manufacturer's protocols. In all cloning experiments, both the linearized DNA vector and the corresponding inserts were purified using the QIAquick Gel Extraction Kit (Qiagen). Subsequently, vectors were dephosphorylated using Antarctic Phosphatase (NEB), and ligations were carried out with T4-DNA ligase (NEB). DH5α-competent cells (custom made) were transformed by heat shock, and cells were grown on LB plates containing selection antibiotics (ampicillin or kanamycin) for 16 h at 37 °C. Single colonies were grown in LB+antibiotic liquid media overnight at 37 °C, and DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen). All constructs were validated by Sanger sequencing (Eurofins genomics) before transfection in HEK293T cells.

Liver was taken from mice at 12 months of age, after 8 months exposure to control, rapamycin, or DR diets. Total RNA was isolated from mouse livers using Trizol (Invitrogen, Carlsbad, CA, USA), cleaned using the Qiagen RNeasy mini RNA cleanup protocol (Qiagen, Valencia, CA, USA), and quantified using a Nanodrop ND-100. Total RNA was then transcribed to cDNA using iScriptTMcDNA reverse transcription kits (1708891; Bio-Rad, Hercules, CA, USA). Reverse transcription products were amplified using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and PCR primers (Integrated DNA Technologies, Coralville, IA, USA). RT–PCR was performed using abi prism sequence detection system instrument and software (Applied Biosystems). Ct data, indicating the number of amplification cycles needed to achieve threshold, were available from six male and six female mice per treatment group. The effect of rapamycin or DR diet was estimated as the difference between Ct for the mutant mouse and Ct for sex-matched littermate controls, with a positive value in cases where expression of the mRNA was increased by diet compared with control animals. Because a change in one unit for Ct reflects a twofold change in mRNA abundance, effect sizes in the figures and tables reflect powers of 2. Thus, an effect size of 5 indicates an increase in mRNA concentration of 2⁵ = 32-fold.