Isopropyl β d 1 thiogalactopyranoside (iptg)

Genetic complementation was achieved by amplifying rsbR1-rsbS-rsbT open reading frames using the primers rsbRST_PstI_V2_F and rsbRST_SalI_V2_R. The obtained amplicon and the IPTG inducible vector, pIMK3, were separately digested using PstI and SalI and ligated afterwards. The resulting plasmid, pDNG7, was transformed into E. coli TOP10 competent cells. The pDNG7 vector was transformed into electrocompetent L. monocytogenes strains and plated into BHI supplemented with Kanamycin 50 μg.ml^-1. Complementation was verified using the primers pIMK3_F and rsbRST_SalI_V2_R on the obtained colonies. Obtained complemented strains were grown at 37°C for 16 h in BHI supplemented or not with 1 mM of IPTG (Sigma) until stationary phase was reached. Strains were challenged in BHI (pH 2.5) acidified with HCl 5 M and samples were taken at 0 and 30 min post challenge, serial diluted in PBS and plated in BHI agar plates. 10 μL of stationary phase cultures of the complemented strains were plated on BHI agar plates supplemented with Congo Red (Sigma) 25 μg.mL^-1 and IPTG 1 mM. Plates were incubated at 37°C then transferred to 30°C for 3 days. At least two biological replicates were made.

RNAi was performed through the generation of inducible shRNA stable cell lines. hTERT-hMSC were transduced with the IPTG inducible lentiviral particles carrying CSNK1A1-specific shRNA (pLKO_IPTG_3XLacO, Sigma-Aldrich, Milan, Italy). Two independent shRNAs (TRCN0000006044, and TRCN0000006042) sequences were chosen. 3 × 10⁴ cells were infected with a multiplicity of infection (MOI) of 4, in the presence of 8 μg/ml polybrene (Sigma-Aldrich, Milan, Italy). 24 h later, the infected medium was replaced with fresh growing medium. Puromycin selection (0.5 μg/ml) was initiated 2 days after transduction. Once a cellular clone was established, to induce CK1α silencing, cells were incubated with 500 μM IPTG (Sigma-Aldrich, Milan, Italy) every 2–3 days for a total of one week. Then, fresh medium without IPTG and puromycin was added for further 48 h. At the end of culture period, cell pellets were collected and analyzed by western blotting to check CK1-α down-regulation and select the more efficient clone. CM was also collected and used for in vitro DC differentiation.

The IPTG/X-gal stock solution: 1.25 g IPTG (Sigma–Aldrich, St. Louis, MO) and 1 g X-gal in 25 mL DMSO (Sigma–Aldrich, St. Louis, MO). Tetracycline (Sigma–Aldrich, St. Louis, MO) stock suspension: 20 mg/mL Tetracycline in 1 mL of 96% ethanol. LB/IPTG/X-gal plates: 1 L LB medium (AppliChem GmbH, Darmstadt, Germany), 15 g/L agar, and 1 mL IPTG/X-gal stock solution. LB/Tet plates: 1 L LB medium, 15 g/L agar and 1 mL Tetracycline stock (15 mg/mL). PEG/NaCl: 20% (w/v) PEG 8000 (Sigma–Aldrich, St. Louis, MO), 2.5 M NaCl. Iodide buffer: 10 mM Tris-HCl (pH = 8), 1 mM EDTA and 4 M NaI (Sigma–Aldrich, St. Louis, MO).

OrthoList RNAi plates were replicated in LB agar, and bacteria were grown overnight at 37 °C. The advantage of growing the bacteria in solid media is the ease of visualising the clones where the bacteria did not grow. If needed, the samples can be kept at 4 °C for 2 days maximum. Next day, we inoculated the RNAi library in 2.2-ml 96-well plates (VWR). Using the pin replicator, we inoculated the bacteria from the LB agar into 1.2 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich). Positive and negative controls were added in the last column of the plate. We cultured the bacteria overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R). In order to have fresh cultures, on the day of sorting, we inoculated 100 μl of the O/N cultures in 900 μl of LB supplemented with ampicillin and tetracycline in deep well plates (VWR) and incubated for 3 h at 37 °C with shaking. We added isopropylthio-β-galactoside (IPTG, 1 mM, Sigma-Aldrich) to the wells to induce the expression of the plasmid for 2 h at 37 °C with shaking. Then, we harvested the cultures by centrifugation (10 min, 3200 g, 4 °C, Eppendorf 5810R) and re-suspended the pellets in 250 μl of S-medium supplemented with carbenicillin (25 μg/ml, Sigma-Aldrich), IPTG (1 mM, Sigma-Aldrich) and cholesterol (5 μg/ml, Sigma-Aldrich).

Plasmids for GST-tagged recombinant proteins were transformed into E. coli BL21. The successfully transformed BL21 cells were cultured in flasks in an incubator shaker and treated with 100 µM IPTG (Sigma-Aldrich) at 18 °C overnight. After IPTG induction, bacteria were collected and resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0) suppled with protease inhibitor (Sigma-Aldrich) and sonicated. Glutathione Agarose (Thermo Fisher Scientific) was added to enrich the GST-tagged protein. The 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0 was added and incubated with agarose for 1 h at room temperature. The eluted protein was collected by centrifuge and saved at −80 °C for further use.

ForshRNA induction, cells were pre-treated with 3mM IPTG (Sigma Aldrich, Cat# I6758) 10-days prior to injection. Xenograft experiments were performed by injecting 3 million human meningiomas cells, either CH157-MN or IOMM-Lee, into the flank of 4–6-week-old NU/NU female mice (Envidigo). To induce plasmid or shRNA expression, mice were treated with or without 200mg/ml doxycycline (Sigma-Aldrich, Cat# D9891) and with or without 10M IPTG in cage water that was changed every 2-3 days. Kaplan-Meyer curves were created by recording deaths at the protocol end of 50% ulcerated tumor or tumor >2000 mm³. Tumors were processed for single-cell dissociation and single-cell RNA sequencing, QPCR, or immunoblotting.

The bacterial strains and plasmids used in this study are listed in Table 1, and the sequences of primers used in this study are listed in Supplementary Table S1. The E. coli strains were grown in Luria–Bertani (LB) broth or on LB agar plates (with 10 g NaCl per liter) at 37°C, except for E. coli carrying pKD46 or pCP20, which were grown at 30°C. Antibiotics and other chemicals were used at the following final concentrations: chloramphenicol, 30 μg/ml; polymyxin B, 2 μg/ml; ampicillin, 100 μg/ml; isopropyl-β-d-thiogalactopyranoside (IPTG), 0.5 mM; and arabinose, 1 mM. A total of 1 mM L-arabinose or 0.5 mM IPTG (Sigma) was used to induce Para or Plac, respectively.