Probenicid

Calcium flux was performed as previously described (24 (link)). Briefly, cells were incubated with 5 mM Indo-1 dye (Invitrogen) and 0.5 mM Probenicid (Sigma-Aldrich) in RPMI without phenol red for 45 min at 37°C. Cells were washed with RPMI, re-suspended in RPMI, and incubated at 37°C for 5 min before Ca²⁺ measurement. A baseline reading was taken for 30 s and cells were stimulated adding CCL21 (100 ng/mL). Samples were analyzed with an LSRFortessa (BD Biosciences) with a UV laser, and data were analyzed with FlowJo software. Calcium increases were monitored as the ratio of Indo-1 (blue) and (violet) emission and displayed as a function of time.

GC-GHRHR cells, transiently transfected with wt-hGH and treated for 24 h with 5 mM butyrate, were plated in half-area clear bottom 96-well plates (Corning, CA, USA) at 75³ cells/well. 24 h later, following two washings with OPTIMEM medium +2.5 mM probenicid (Sigma-Aldrich, Buchs, Switzerland), cells were incubated with 2 µg/mL Fluo-4 AM (Invitrogen, LifeTechnologies, Basel, Switzerland) and Pluronic F-127 0.025% (w/v) (Invitrogen, LifeTechnologies, Basel, Switzerland) for 30 min in dark at 37°C. After washing twice with OPTIMEM medium, cell measurement was performed on a Synergy-4 instrument (BioTek, Highland Park, VT, USA) with an excitation band of 485/20 nm and fluorescence was measured at 528/20 nm. Baseline signal (F₀) was recorded during 5 min before the addition of each stimulus. Subsequently, continuous fluorescence measurements were performed for 20 to 30 min. Ionomycin (Calbiochem, San Diego, CA, USA) stimulation was used as a positive control. Results are shown as F/F₀ ratios after background subtraction, where F was the fluorescence signal intensity and F₀ was the baseline as calculated by averaging the ten frames before stimulus application.
For [Ca²⁺]_i measurements in siRNA-treated cells, cells were further transfected with wt-hGH 48 h post-siRNA transfection and treated with 5 mM butyrate. After 24 h cells were seeded and treated as mentioned above.

Astrocyte cells (C6, rat astrocyte cell line) and endothelial cells (bEnd.3, mouse endothelial cell line) were from ATCC (Manassas, VA, USA). Cell culture inserts for 24-well plates (transparent PET membrane transwells, 0.4 µm pore size) were obtained from Dominique Dutscher (Alsace, France). Imaging Plate FC, 24-well plates, TC-surface was purchased from Ozyme (Saint Quentin Yvelines, France). The EVOM voltohmmeter system was from World Precision Instruments (Hertfordshire, UK). ZO-1 antibodies were from Life Technologies (cat 40-2200; Saint Aubin, France) and claudin-5 antibodies were from ABCAM (ab15106; Paris, France). P-gp and MRP-1 antibodies were from GeneTex (GTX23364; San Antonio, TX, USA) and Santa Cruz Biotechnology (sc-13960; Dallas, TX, USA), respectively. Standard protein and all compounds for Ringer HEPES buffer, BCECF-AM, probenicid, verapamil, sodium fluorescein (Na-Fl), were from Sigma-Aldrich (St. Quentin Fallavier, France). Rhodamine-123 was from Thermo Fisher Scientific (Eugene, OR, USA). Hydralazine, MTT kit and Dulbecco’s Modified Eagle’s Medium (DMEM) were also from Sigma-Aldrich. Antibodies and reagents for the detection of HIF-1α were products of R&D systems (Lille, France). YC-1 was purchased from VWR International (Fontenay-sous-Bois, France).