Stat5 in 1

Mouse naive CD8⁺ T cells were negatively selected by EasySep Mouse Naive CD8⁺ T Cell Isolation Kit (STEMCELL). Isolated naive CD8⁺ T cells were cultured in RPMI-1640 medium with 10% FBS, 1% penicillin, and 100 μg/mL streptomycin in flat-bottom plates precoated with anti-CD3 (2 μg/mL; clone 145-2C11; eBioscience) and anti-CD28 (2 μg/mL; clone 37.51; eBioscience) for 2 days, unless otherwise stated. For small molecular inhibitor treatment, Stattic (Selleck, S7024), SH-4-54 (Selleck, S7337), STAT5-IN-1 (Selleck, S6784), or 17-AAG (Selleck, S1141) was added to culture medium and cells were cultured for indicated times.

Atherosclerotic plaque samples obtained during carotid endarterectomy from 4-10 symptomatic patients (Supplemental Table 2) were collected in the Maastricht Pathology Tissue Collection (MPTC) in line with the Dutch Code for Proper Secondary Use of Human Tissue (http://www.fmwv.nl) and the local Medical Ethical Committee (protocol number 16-4-181). This study conforms to the Declaration of Helsinki. The endarterectomy specimens were cut into 2 mm thick sections. Subsequently, the sections were cultured in RPMI1640 (Thermofisher) supplemented with 0.1% FCS and 1% PenStrep (Gibco) and cultured in a controlled environment (37°C, 5% CO₂) for 24 hours in presence of the general STAT5 inhibitor STAT5-in-1 (300 µM, Selleck Chemicals) or the STAT5A-specific inhibitor POM-CHF-Stafia-1 (50 µM, Supplemental Figure 1) (25 (link)). Control sections and inhibitor treated sections were alternating. Control sections were treated with DMSO concentration equal to the inhibitors. A multiplex ELISA was performed on the plaque-conditioned medium as described above.

To evaluate the effects of vitamin A, vitamin D, signal transducers and activators of transcription 5 (STAT5)-inhibitor STAT5-IN-1, or p38 mitogen-activated protein kinase (p38 MAPK)-inhibitor SB203 580 (5 µM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany) on organoid cell viability, an MTT reduction assay was performed. For this purpose, organoids were incubated with calcitriol (50 nM, 100 nM, 200 nM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany), or with retinoic acid (10 µM, 100 µM, 200 µM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany), or with STAT5-IN-1 (5 µM, 0.5 mM, 1 mM, solved in DMSO; Selleckchem, Planegg, Germany), or with SB203 580 (5 µM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany), or with a corresponding amount of DMSO as control for 30 h. Then, MTT solution (500 µg/mL; solved in PBSO; Merck, Darmstadt, Germany) was added to organoids to a final concentration of 500 µg/mL. After incubation for 1 h at 37 °C, 5% CO₂, CCM was discarded and cells were incubated with 20 µL of SDS (for 1 h at 37 °C, 5% CO₂) to dissolve Matrigel. Finally, 100 µL of DMSO was added (for 1 h at 37 °C, 5% CO₂) to dissolve reduced MTT, and the optical density was measured at 562 nm on a microplate absorbance reader (BioTek Instruments, Winooski, VT, USA).