Facsdiva software v6

Forty-eight hours after transfection, cells were fixed with Cytofix/Cytoperm™ (BD Biosciences) and incubated with anti-Beclin 1 (1:200; Abcam, Cambridge, UK) or anti-Fibulin 5 (1:200; Biorbyt, Cambridge, UK) antibodies for 40 min. Next, the cells were incubated with secondary antibodies, as appropriate, for 30 min. The transfected cells expressing GFP protein encoded by the pSELECT-GFPzeo backbone and protein of interest were analyzed with a BD FACS Canto II Cytometer and BD FACS Diva Software v. 6.1.3 (BD Biosciences, San Jose, CA, USA).

The ALDEFLUOR assay kit (Stem Cell Technologies) was used to detect the activity of ALDH. Single cells isolated from cell lines or tumor samples were mixed in ALDEFLUOR assay buffer (1.5 µM) containing the ALDH substrate BODIPY amino acetaldehyde (BAAA) and incubated for 1 h at 37 °C. Diethylamino benzaldehyde (DEAB), was used as a negative control (tenfold molar excess). BD FACS Diva software V6.1.3 (BD Biosciences) or Flow Jo software (Tree Star) were used to analyze flow cytometry data.

Peripheral blood was collected before surgery into K₃EDTA BD Vacutainer tubes (BD Biosciences, San Jose, CA, USA) and kept at room temperature until preparation for flow cytometry within 4 h. In total, 50 µL of whole blood was added to each flow-cytometry tube with appropriate amounts of fluorochrome-labeled mAbs (as determined after titration of the dose recommended by the manufacturers). The following antibodies were used in this study: anti-CD14^APC (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD16^FITC (ThermoFisher, Waltham, MA, USA). Data were collected on a BD FACSCANTO II flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with the BD FACSDiva Software v6.1.3 (BD Biosciences, San Jose, CA, USA).

For STAT1 expression analysis: 2.0 × 10⁶ cells were fixed in BD Phosflow^™ Lyse/Fix Buffer 1× (BD Biosciences, Le‐Pont‐de‐Claix, France) for 10 min. at 37°C and further permeabilized using BD Phosflow^™ PERM buffer III (BD Biosciences) following the instructions of the manufacturer. Cells were labelled using the following antibodies: Alexa Fluor^® 647 Mouse Anti‐STAT1 (N‐terminus) (#558560) or Alexa Fluor^® 647 Mouse IgG1 κ control isotype (#557783; BD Biosciences). Data were collected with a Becton Dickinson FACS Canto II (BD Biosciences) and analysed with BD FACS DIVA Software V6.1.3 (BD Biosciences). For cell cycle analysis, 1.0 × 10⁶ cells were exposed to 10 μM MNNG for 1 hr. Cells were collected at the indicated times after treatment. After fixation in ice‐cold 70% ethanol, cells were recovered by centrifugation and further incubated with RNAse A (1 μg/ml; Thermo Fisher) for 30 min. at room temperature. After three washes, 20 μg/ml propidium iodide (Sigma‐Aldrich) was added and incubated at room temperature in the dark for 45 min. prior to analysis. Data were analysed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

For the initial sort, 100 μL of the library was inoculated into 25 mL of medium with inducers and grown overnight for 18–24 h. The next day, 10 μL of the culture was diluted into 10 mL 1× phosphate-buffered saline and then passed into the cytometer. Cell sorting was performed on a FACS Aria IIu cytometer (BD-Biosciences) with BD FACS Diva Software v6.1.3, through the red fluorescence channel (excitation 561 nm, emission 610/20 nm), under the Purity Mode. Cells were first gated on irregularly shaped FSC-A and SSC-A gates to exclude non-cellular materials, and then gated on boundaries defined by the previous libraries with or without induction. Gate sizes and positions were tailored to individual experiments. See Supplementary Fig. 28b for example gating strategies. Typically, 0.5–1 million gated events were collected into a 15 mL conical tube with 5 mL of LB supplemented with 1 % d-Glucose (10117, VWR) and without antibiotics. Collected cells were recovered for 2 h at 37 °C with 160 rpm shaking. Then, the volume was topped to 15 mL using LB with the next set of inducers and grown overnight for 16–18 h for the next sorting experiment. After the final cell sorting, the overnight culture was diluted and plated onto LB agar to obtain single colonies for strain isolation.