Rapamycin

Peripheral blood mononuclear cells (PBMCs) (Cellero, Wilmington, MA, USA, catalog#1001, Lot# 4914OC20) were seeded into a poly-L-ornithine coated 96-well plate at 40,000 cells per well and cultured in a 1:1 mixture of RPMI1640 medium (Gibco, catalog# 11875093) containing 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA, catalog# S11550) and 1% GlutaMAX™ and MEM α medium supplemented with 5% hPL and 1% GlutaMAX™. Phytohemagglutinin (PHA-P, PHA) (Sigma, catalog# L1668-5MG) at 10 µg/mL, rapamycin (InvivoGen, San Diego, CA, USA, catalog# tlrl-rap) at 10 µg/mL, and CCM from naïve MSCs or eMSCs-IL10 were added to the respective groups. For the groups with the IL-10 receptor α antibody (IL10 Ra AB) (R&D Systems, Minneapolis, MN, USA, catalog# MAB274), PBMCs were incubated with 80 µg/mL of IL10 Ra AB for 1.5 h prior to adding other treatments, PHA, and CCM-IL10 (100 ng/mL). After 3-day incubation, the cell viability was determined using the CellTiter-Glo^® Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA, catalog# G7571), which is based on ATP quantification. The data were normalized to the PBMC group stimulated with PHA. One-way ANOVA was used for statistical analysis (n = 4).

Eight- to ten-week-old male C57BL/6 J mice purchased from Harlan Laboratories (Indianapolis, IN, USA) were adapted to the environment for one week before the study. The inducible liver IR knockout (iLIRKO) mice were described previously^{21 (link)}. Fifteen-week-old male mice were studied 2 weeks after 3 consecutive injections with tamoxifen (1.5 mg/mouse). All mice were housed in colony cages with a 12 h/12 h light/dark cycle in a temperature-controlled environment (lights off at 3:00 pm). Mice had free access to water and regular diet (65% carbohydrate, 11% fat and 24% protein). For “fasting” and “refed” conditions, mice were either fasted for 24 h, or refed overnight on a regular diet with 20% glucose in tap water after 24 h fasting. For insulin signaling inhibitors experiments, mice were injected twice (18 hours and 2 hours) before sacrifice with wortmannin (2 mg/kg, InVivoGen, CA, USA) or rapamycin (4.5 mg/kg, InVivoGen). Livers were frozen in liquid nitrogen and kept at −80 °C until use. Plasma samples were frozen and stored at −80 °C until use. All procedures were carried out according to the French guidelines for the care and use of experimental animals. All animal studies were approved by the “Direction départementale des services vétérinaires de Paris”.

Aldrin, BDE28, PCB153 and PFOA were purchased from Sigma^® (Darmstadt, Germany) and then diluted in ethanol to a 10⁻³ M concentration stock, except for PCB153 which was diluted to a 10⁻⁵ M concentration stock. LY294002, a PI3K inhibitor, and rapamycin, a mTOR inhibitor, were, respectively, purchased from InVivogen^® (Toulouse, France) and Sigma^®.

LA-4 cells were pre-incubated with the indicated amounts of either the mTOR inhibitor rapamycin (Invivogen, Toulouse, France), MAPK-inhibitors U0126 (MEK1/2 MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands), SP600125 (SAP/JNK MAPK inhibitor, Invivogen), SB-202190 (p38α/β MAPK inhibitor, Invivogen), the NFκB- and MAPK-inhibitor dexamethasone (Invivogen), the IKK-β-inhibitor TPCA-1 (Abcam, Berlin, Germany), the inhibitor of actin polymerization cytochalasin A (Sigma-Aldrich), the inhibitor of endosomal acidification chloroquine (InvivoGen), or the COX2-inhibitor NS-398 (Sigma-Aldrich, Steinheim, Germany) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. For analyzing cell viability, LA-4 cells were treated as indicated and stained for dead cells using the fixable viability dye eFlour450 (#65-0865-14, eBioscience) and measured by FACS. Data were analyzed using FlowJo V.7 (Treestar Inc., Ashland, OR, USA) and GraphPad PRISM (GraphPad Software, San Diego, CA, USA).

Ovaries were placed in cryopreservative medium containing Leibovitz L-15 medium supplemented with 10% FBS, 10% dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) and 0.1 M sucrose. After equilibration in cryopreservation medium for 30 min at 4 °C, ovaries were placed in cryovial tubes (Simport, Montreal, Quebec, Canada) and subsequently cooled in a programmable freezer (CL-8800i System; CryoLogic, Mulgrave, Victoria, Australia) as described previously [55 (link)] and stored in liquid nitrogen. For slow freezing with inhibitors, LY294002 (25 µM, InvivoGen, Toulouse, France) or Rapamycin (1 µM, InvivoGen, Toulouse, France) were added in the transport and cryopreservation media.
For thawing, cryovials were incubated at room temperature for 2 min and thawed by rapid immersion at 37 °C in a water bath. To remove cryoprotectants, ovaries were washed three times for 5 min at 37 °C in Leibovitz L-15 medium.

Mice were intraperitoneally (i.p.) immunized with 50 µg TNP₃₆-Ficoll T-cell-independent antigen (Biosearch Technologies) or with 100 µg TNP₁₈-KLH T-cell-dependent antigen (Biosearch Technologies) with adjuvant (Sigma) at day 0. Serum samples were collected from the tail vein (day 0) or by cardiac puncture (day 7 and day 10, respectively). Rapamycin (InvivoGen) was diluted in 100-µl phosphate buffer solution (PBS) with vehicle including 0.25% PEG and 0.25% Tween 80 (Sigma) and injected i.p. at a dose of 1 mg/kg. Injections were given daily, starting 1 day before immunization and ending 7 or 10 days after immunization, respectively. Control mice were injected with the same volume of PBS and vehicle.