Hygromycin

Flp‐In HEK293T‐REx cells were transfected with pcDNA5/FRT/TO AFG3L2^E408FLAG. After transfection cells were selected with 100 μg/ml hygromycin (Roth, Cat# CP13.4) for 7 days. Single colonies were selected and expression of the gene of interest was induced with 1 μg/ml tetracycline (Roth, Cat# 0237.1) for 24 h before testing expression via immunoblotting.

Si28 iPSCs were infected with the lentivirus described in Fig. 2A (also see Supplementary Material). In brief, infected cells underwent hygromycin (Carl Roth, Karsruhe, Germany) selection followed by manual picking and expansion of the colonies. Stocks of the clones were cryopreserved in 90% FBS and 10% dimethyl sulfoxide. Short tandem repeat (STR) DNA typing (described in detail in Ref. ^{36 (link)}) was performed for cell line authentication. To evaluate the clone’s NGN1 expression properties, iPSCs were seeded as single cells in E8 medium and exposed to doxycycline (2 µg/mL) for up to 5 days (Fig. 2B).

Stable GLI reporter AML cell lines were transduced by lentiviral constructs containing the firefly luciferase gene under the control of GLI transcriptional response elements and as internal control the renilla luciferase gene under the control of CMV promoter elements (Cignal™ Lenti Reporters, S-6030L, Qiagen, Venlo, The Netherlands) followed by puromycin (P7255, Sigma-Aldrich, St. Louis, MO, USA) and hygromycin (1287.1, Carl Roth GmbH, Karlsruhe, Germany) selection. Stable GLI reporter cells were treated with MBZ, GANT-61, PU-H71, VER-155008 or a solvent control, with the GLI promoter activity measured after 24 h using the Dual-GLO Luciferase Assay Kit (E2940, Promega, Madison, WI, USA) and the Infinite F200 PRO reader (Tecan, Männedorf, Switzerland). The firefly luciferase-mediated GLI promoter activity was normalized to the renilla luciferase-mediated CMV promoter activity.

In order to re-isolate the selected BCG-GFP (SEL) in vitro, LTI macrophages were lysed in sterile water and plated onto Middlebrook 7H10 agar medium supplemented with 10% OADC. The plates were incubated at 37°C in 5% CO₂ atmosphere. After 3 or 4 weeks, some of the grown colonies were transferred to Middlebrook 7H9 medium supplemented with 10% OADC, 0.2% glycerol, 0.05% Tween 80 (complete medium) and 50 μg ml⁻¹ hygromycin (Roth, Germany). SEL was incubated for about 2 weeks at 37°C in a 5% CO₂ atmosphere. At this point, several aliquots of the SEL were stored at −80°C. In order to maintain the features of the SEL bacteria, new SEL bacteria were thawed and used for all the in vitro experiments (passage 1, 2 or 3).

Mycobacterium tuberculosis H37Rv ATCC 27294 was used as a parental strain for genetic manipulations. Escherichia coli HB101 served as a host for molecular cloning procedures. Middlebrook 7H10 agar (Becton Dickinson, Franklin Lakes, USA) containing 0.5% glycerol (Applichem, Darmstadt, Germany), 10% OADC (Becton Dickinson, Franklin Lakes, USA), and 0.05% Tyloxapol (Sigma-Aldrich, St. Louis, USA) for 7H9 broth if required supplemented with 50 µg/ml hygromycin (Carl Roth, Karlsruhe, Germany) was used to cultivate Mtb strains, whereas Luria Bertani (LB) medium (Carl Roth, Karlsruhe, Germany) was used to cultivate E. coli. LB supplemented with Ampicillin (Carl Roth, Karlsruhe, Germany) was used as a selective medium for E. coli. For animal infection experiments Mtb stocks were prepared as follows. Bacteria were grown in 7H9 broth to mid-exponential growth phase and aliquots frozen at −80 °C. Titers of viable bacteria were determined after thawing by plating serial dilutions of the aliquots on 7H10 agar plates. Single cell suspensions were prepared by pushing the bacteria 10 times through a Microlance 3 26-gauge needle (Becton Dickinson, Franklin Lakes, USA). These suspensions were used for aerosol infection.

U2OS 2-6-3 CLTon cells38 (link) were kindly provided by Dr Supriya Prasanth (University of Illinois, IL, USA) and maintained in DMEM (high glucose+GlutaMax) (GIBCO/Invitrogen, Darmstadt, Germany) supplemented with 10% (v/v) TET system approved fetal bovine serum (Clontech, Saint-Germain-en-Laye, France), 50 μg ml⁻¹ hygromycin (Carl Roth, Karlsruhe, Germany) and 200 μg ml⁻¹ G418 (Clontech, Saint-Germain-en-Laye, France). For fluorescence microscopy, cells were grown on coverslips in a six-well plate without antibiotics. 500 ng plasmid DNA was transfected using Lipofectamine LTX and Plus reagent (Invitrogen, Darmstadt, Germany). 20 h post transfection, cells were treated with 1 μg ml⁻¹ doxycycline for 24 h for induction of rtTA expression. Cells were washed twice with PBS and fixed with 3.7% (v/v) formaldehyde/PBS for 10 min at RT. Cells were permeabilized with 0.5% (v/v) Triton-X100 (PBS) for 5 min at RT and incubated with 200 ng ml⁻¹ DAPI (PBS) for 5–10 min. Cells were mounted in Vectashield (Vectorlabs, CA, USA). Images were acquired using a Leica SP5 confocal microscope with a 63 × oil immersion lens (Leica Mikrosysteme, Wetzlar, Germany). P values (according to Student's t-test) were calculated using GraphPad QuickCals (GraphPad, CA, USA).

The bacterial strains used in this study are listed in Table 1. The E. coli strains were grown in Luria–Bertani (LB) broth medium. When required, antibiotics (Carl Roth, Karlsruhe, Germany; Sigma-Aldrich, St. Louis, MO, USA) were added to the cultures at the following concentrations: 75 μg mL⁻¹ ampicillin, 50 μg mL⁻¹ kanamycin, 50 or 120 μg mL⁻¹ hygromycin, and 50 μg mL⁻¹ apramycin (Carl Roth, Karlsruhe, Germany; Sigma-Aldrich, St. Louis, MO, USA). E. coli GB05-red [21 (link)] was employed in Red/ET recombineering experiments [22 (link)].
For conjugation, the Streptomyces albus J1074, del9 and del10 [19 (link)] strains were grown on oatmeal or mannitol soy (MS) agar [23 ] for sporulation.