Crl 1469

Minimum inhibition concentration (MIC) was determined using the microbroth dilution method according to guidelines of the Clinical Laboratory Standards Institute (CLSI) with slight modifications. Assays were carried out with C. albicans ATCC^® 90028™ at 2.5 × 10⁵ cells mL⁻¹, A. fumigatus ATCC^® 46645™ at 2.5 × 10⁴ spores mL⁻¹ and both S. aureus Rosenbach ATCC^® 25923™ and Escherichia coli (ATCC^® 25922™) at 5 × 10⁵ cells mL⁻¹. The cells were incubated with the compounds at 35 °C for 24 h (C. albicans) and 48 h (A. fumigatus); and 37 °C for 24 h for both bacteria. The effect of the compounds on bacterial growth was evaluated by measuring the optical density at 600 nm using a microplate reader. Mammalian cell cytotoxicity was assessed with A549 human lung carcinoma cells (ATCC^® CCL-185™) seeded at 1500 cells well⁻¹, while both PANC-1 human pancreatic carcinoma cells (ATCC^® CRL-1469™) and HepG2 human hepatocellular carcinoma cells (ATCC^® HB-8065™) which were both seeded at 2500 cells well⁻¹. These cells were treated with the compounds for 72 h at 37 °C in the presence of 5% CO₂. PrestoBlue™ cell viability reagent (Life Technologies) was used to assess the cytotoxic effect of the compounds. Microplates were incubated with this dye for 2 h before measuring the fluorescence at excitation 560 nm and emission 590 nm. All assays were performed in triplicate on two different test runs.

PANC-1 cells, an epithelioid carcinoma cell line derived from the human pancreas [30 (link)], were provided by ATCC (frozen, CRL-1469, tissue: Pancreas; Duct) from the human cell culture collection (https://www.atcc.org/ accessed on 1 September 2020).

WT MIA PaCa-2 and PANC-1 are immortalized epithelial cellular line of human pancreatic carcinoma and they were purchased from ATCC (ATCC CRL-1420 and ATCC CRL-1469, respectively, for MIA PaCa-2 and PANC-1 cells, Manassas, VA, USA). ANXA1 knock-out (KO) MIA PaCa-2 cells were obtained from the WT cells through the CRISPR/Cas9 genome editing system, as reported in [14 (link)], and kept in selection by 700 µg/mL neomycin (Euroclone; Milan, Italy). The cells were cultured as reported in [39 (link)] and maintained at 37 °C in a 5% CO₂-95% air humidified atmosphere.

PANC-1 cells were purchased from ATCC (CRL-1469, lot#6778038) and maintained in D-MEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Confluent cells were stimulated by replacing the culture medium with a warmed medium containing 3 μM of CHIR-99021 (Chemscene LLC, CS-0181) and 30 μM of ICG-001 (synthesized in house) or the same volume of DMSO and cultured for 24 hours for ChIP-seq, ATAC-seq, and RNA-seq.

The cell lines PANC-1 (CRL-1469™, pancreatic), PANC0403 (CRL-2555™, pancreatic) and 4T1 (CRL-2539™, breast) were obtained from ATCC®. A375Ppuro and A375Pβ6puro cell lines were created using the human melanoma cell line A375P (CRL-3224™, melanoma), which was infected with pBabe retroviruses encoding puromycin resistance alone or in combination with cDNA for human β6, as previously reported [12] (link). The A375Ppuro and A375Pβ6 cell lines were a kind gift from Prof. John Marshall (QMUL). The A375Pβ6 cell line was subsequently transfected with firefly luciferase (luc) using an SFG retroviral vector whereby luc was co-expressed with dsTomato red fluorescent protein. Transduced cells were then flow sorted for red fluorescence to obtain a pure A375Pβ6-luc cell line [24] (link). All cell lines were maintained at 37 °C, 5% CO₂ and 5% relative humidity. Advanced RPMI (PANC-1, PANC0403, 4T1) or DMEM media (A375Ppuro, A375Pβ6puro) were used, both of these were supplemented with 10% FBS, 1% GlutaMAX™ and 1% Penicillin/Streptomycin.

The human pancreatic adenocarcinoma AsPC-1 and Capan-2 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Panc-1 and MIA Paca-2 cell lines were cultured in DMEM medium supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. The cells were cultured and maintained as previously described [21 (link)]. We obtained AsPC-1 cells (ATCC® CRL-1682™) in 2010, MIA Paca-2 (ATCC® CRL-1420™) cells and Panc-1 (ATCC® CRL-1469™) cells in 2016 and Capan-2 cells (ATCC® HTB-80™) in 2019. All cell lines were obtained from ATCC (Manassas, VA, USA). After obtaining cells from ATCC, cells were frozen at low passage (p3-p5), and then thawed and used at passage 10 or lower. These cell lines were tested for mycoplasma contamination at regular intervals. we did not authenticate these cell lines.

HEK293T and HEK293T-NTSR1 cell lines were purchased from trenzyme (Trenzyme, #1564). HEK293T-NTSR1 stable cell line was generated with the transduction into the HEK293T cells of an optimized-mutated sequence of NTSR1 that translated into the normal amino acid sequence of NTSR1. PANC1 (ATCC, CRL-1469) and HT29 (ATCC, HTB38) were purchased from ATCC.