Doxycycline hydrochloride

To characterize RNA exporter expression dynamics during the population dynamics experiment, as shown in Figure S5E, monoclonal inducible RNA exporter-expressing cells (cFH14.1) were plated on a 6-well plate, induced with doxycycline hydrochloride (Sigma) at 1 ng/μL for 2 days, and purified GFP+ cell populations were sorted into wells of a 96-well plate. For the zero timepoint, the sorted cells were immediately analyzed. For subsequent timepoints at days 1, 3, 5, and 7, the cells were lifted from the plate using 50 μL of Trypsin-EDTA (ThermoFisher), resuspended by addition of 150 μL of media, passed through a 35 μm filter, and analyzed on a CytoFLEX S flow cytometer (Beckman Coulter). Data were analyzed by using FlowJo software (10.8.1) to gate for live single cells based on forward and side scatter, then calculating the fraction of cells expressing GFP at each timepoint.

Collagen type I extracted from bovine skin was supplied by National Research & Development Institute for Textiles and Leather, Bucharest, Romania. The antimicrobial drugs oxytetracycline hydrochloride and doxycycline hydrochloride were purchased from Sigma Chemical Co. (Saint-Louis, MO, USA), and glutaraldehyde was used as the cross-linking agent and purchased from Merck (Darmstadt, Germany). For pH adjustment, 1 M of NaOH solution of analytical grade was used.
Collagen supports preparation, in the form of sponges, was based on a freeze-drying process [30 (link)] of homogenous mixtures containing CG, OTC, or DXC and GA. CG 1% with pH adjusted to 7.4 was mixed with a water solution of OTC or DXC 1 g/L for 30 min, and after another pH adjustment to 7.4, different concentrations of GA reported to dry CG and OTC or DXC substances were added and stirred for 10 min. The resultant mixtures were then freeze-dried. This process consists of three steps as follows: “Freezing” at −55 °C for 10 min, “Main drying” at 55 °C for 15 h, where ~90% of water is removed, and “Final drying” at 40 °C for 10 min. All the collagen sponges were packed in polyethylene bags and sterilized for 5 min on each side at 254 nm using a Vilber-Lourmat equipment.
The composition of the collagen sponges is shown in Table 1.

The HEK293 and NIH3T3 [50 (link)–52 (link)], MCF-10A, and MDA-MB-231 [9 (link), 14 ], and the KLF8-expressing Tet-on MCF-10A (10A-iK8) and the KLF8 shRNA-expressing Tet-on MDA-MB-231 (231-K8ikd) cell lines were described elsewhere [31 (link)]. These cells were maintained in DMEM/F-12 or DMEM with 10% fetal bovine serum or calf serum. The 10A-iK8-miR141 and 231-K8ikd-miR141 were generated by infecting 10A-iK8 and 231-K8ikd cells with viruses derived from pLenti 4.1 miR141 followed by puromycin selection. The 10A-iK8-miR141sponge and 231-K8ikd-miR141sponge cell lines were generated by infecting the cells with viruses derived from the pBABE-puro-mcherry-miR141sponge plasmid followed by puromycin selection. The cell lines were maintained under U (in the absence of doxycycline) or I (in the presence of doxycycline) conditions depending on the experimental need. Doxycycline hydrochloride was purchased from Sigma-Aldrich Corp. (D3072, St. Louis, MO, USA).

Differentiation into i³Neurons was as previously described, with slight modifications.³⁰ (link) Briefly, on day 0 iPSCs were dissociated into single cells using StemPro Accutase (Thermo Fisher Scientific) and seeded at a density of 150,000 cells/cm² on Matrigel-coated culture dishes in Induction Medium (IM) composed of DMEM/F-12, 1× N-2 Supplement, 1× MEM Non-Essential Amino Acids Solution, 1× GlutaMAX Supplement (all Thermo Fisher Scientific), 10 μM Y-27632, and 2 μg/mL doxycycline hydrochloride (Sigma-Aldrich). Pre-differentiated cells were maintained in IM for 3 days with daily medium changes. After the 3-day differentiation period, cells were dissociated with StemPro Accutase and seeded at 5 × 10⁴ cells/cm² onto culture plates coated with 0.1 mg/mL poly-L-ornithine (Sigma-Aldrich). Cells were maintained in Cortical Neuron Culture Medium, composed of BrainPhys Neuronal Medium (STEMCELL Technologies), 1× B-27 Supplement (Thermo Fisher Scientific), 10 ng/mL BDNF (PeproTech), 10 ng/mL NT-3 (PeproTech), and 1 μg/mL mouse Laminin (Thermo Fisher Scientific) with half media changes carried out every 3–4 days.

For genetic labeling of blood vessels, Rosa26-tdTomato mice (B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) (Madisen et al., 2010 (link)) were mated with mice expressing Cre recombinase under the control of the endothelium-specific promoter VE-Cadherin (VECad-Cre; Jackson Laboratories).
To switch Vegf overexpression on and off in the developing skeleton, we used a triple transgenic mouse line. We crossed heterozygous Col1a-Cre mice carrying a promoter specific to osteoblasts (Dacquin et al., 2002 (link)) with mice heterozygous for reverse tetracycline transactivator (rtTA) (Belteki et al., 2005 (link)) and tetracycline-responsive element (tetO)-driven transgene (tetO-Vegf) (Dor et al., 2002 (link)). Overexpression was induced by adding 200 μg/ml doxycycline hydrochloride (Sigma, D9891) in 3% sucrose to the drinking water of pregnant females from embryonic day (E) 13.5 until E15.5-E18.5. As a control, wild-type littermates were used. All animal experiments were pre-approved and supervised by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute.
In all timed pregnancies, plug date was defined as E0.5. For harvesting of embryos, timed-pregnant female mice were sacrificed by cervical dislocation. Tail genomic DNA was used for genotyping by PCR.