Vi cell

Kinetic cAMP assays were performed in low-density GIPR and GLP-1R cell clones transfected with the Glosensor 22F vector (Promega) (53 (link)). Adherent cells were transiently transfected with Glosensor at 7.5 μg DNA/30 μL Fugene-6 per 150 cm² flask, according to manufacturer’s directions. After 48 hours, cells were removed from flasks using enzyme-free dissociation buffer (13151014, Thermo Fisher Scientific), followed by trituration, brief centrifugation (500g, 5 minutes, room temperature) and filtration (40 μm cell strainer, Thermo Fisher Scientific). Cell viability was quantified with a ViCell (Beckman Coulter) and was typically > 85%. Cells were resuspended at ambient temperature in CO₂-free media (18045088, Thermo Fisher Scientific) containing 0.1 %(v/v) casein (C4765, MilliporeSigma) and 2%(w/v) Glosensor detection reagent (Promega). A total of 18,000 cells per well was plated in 180 μL in solid-bottom, white 96-well plates (3917, Corning). Luminescence was quantified in kinetic mode using a temperature-controlled (22°C) EnVision (PerkinElmer) using standard luminescence settings. Cells were equilibrated for 5–20 minutes, and then 20 μL of 10× ligand was added and a luminescence time course was collected. Ligand (10×) was titrated by manual serial dilution in DMSO followed by step-down into assay buffer.

In vitro function was assessed by measuring in vitro glucose-stimulated insulin secretion17 (link). Clusters were washed twice in Krebs buffer (krb), preincubated for 2 h in krb containing 2 mM glucose (low glucose) at 37  °C and washed once. Clusters were then challenged with three sequential treatments of alternating low-glucose krb and high-glucose krb containing 20 mM glucose (sx total glucose treatments), followed by treatment with low-glucose krb containing 30 mM KCl. Each treatment lasted 30 min, after which 100 μl of supernatant was collected and human insulin quantified using the Human Ultrasensitive Insulin ELISA (ALPCO Diagnostics; 80-INSHUU-E01.1. Human insulin measurements were normalized by viable cell counts that were acquired by dispersing clusters with TrypLE Expression and counted using a ViCell (Beckman Coulter). The formulation for krb buffer is seen in Supplementary Table 7. In a subset of experiments, cells were pre-cultured with an anti-diabetic drug for 24 h in stage 6 media and that same anti-diabetic drug was also included in the krb buffer: Tolbutamide (100 μM; Sigma; T0891), LY2608204 (1 μM; Selleck; S2155) and Liraglutide (1 μM; Bachem; H-6724.0001).

Cells were seeded in 12-well plates with approximately 0.5 × 10⁵ cells per well and allowed to attach overnight at 37°C, 5% CO₂. Cells were then treated with GCB or GCB coupled with HCQ; cells with no treatment served as controls. Cells were trypsinized and viability was read using a cell counter (Vi-cell, Beckman Coulter). All experiments were performed three times. The incubation time and drug concentrations are indicated in each figure.

PC-3 cells were seeded on small Petri dishes d = 35 mm at a concentration of 3 × 10⁵ cells in 5 mL of DMEM medium for 24 p for adhesion at 5%, CO₂, 37 °C. Then, 50 μL of Kuk solution at various concentrations was added to the cells and placed in a CO2 incubator for 48 h. Then, the cells were treated with trypsin (0.25% trypsin in 1 mM EDTA solution) and washed with PBS by centrifugation for 5 min, 1500 rpm, and resuspended; the number of living and dead cells was counted using an automated Beckman Coulter Vi-CELL instrument (Beckman Coulter, Krefeld, Germany) and seeded into 6-well plates at a concentration of 100 live cells in 3 mL of DMEM medium. After 10 days of incubation, the medium was taken, the cells were fixed with 1 mL of 100% methanol for 25 min, washed with PBS and dried, and 1 mL of Giemsa solution was added, and then they were incubated for 25 min at room temperature and washed with dH₂O [36 (link)]. The cells were then air dried and the number of colonies was counted. Results were expressed as the number of colonies grown per well.

The in vitro effect of the drugs on cell viability and proliferation was evaluated using trypan blue exclusion assay as described before with minor modifications [49 (link)]. In brief, cells (0.2 × 10⁶ cells/well) were seeded in 12-well plates, incubated overnight, the media was replaced with fresh media (1 mL/well) and treated with the drugs for 48 h. Cells were harvested by trypsination and the viability was measured using trypan blue staining and Beckman Coulter Vi-CELL (Beckman Coulter, Krefeld, Germany).