Rhegf

HeLa S3, HEK293, and Cos7 cells were cultured in DMEM containing 10% FBS. These cell lines were obtained from the American Type Culture Collection or the Japanese Collection of Research Bioresources and regularly tested for Mycoplasma contamination. The antibodies and suppliers were as follows: anti-pY1068-EGFR [cat. no ab32430, Abcam or cat. no 2234, Cell Signaling Technology (CST)]; anti-EGFR (cat. no MI-12-1, MBL); anti-phospho-ERK (cat. no 9106, CST); anti-ERK (cat. no sc-94, Santa Cruz Biotechnology); anti-GFP (598, MBL); anti-Flag (M2, cat. no F1804, Sigma or FLA-1, cat. no M-185-3L, MBL); anti-V5 (cat. no 46-0705, Invitrogen); anti-PTP1B (cat. no ab252928, Abcam); and anti-annexin A1 (cat. no ab135256, Abcam). Affinity-purified rabbit antibodies against pY944-LRRK1 were produced according to a previously described method (Ishikawa et al., 2012 (link)). Rh–EGF and EGF were purchased from Sigma. A647–EGF was purchased from Thermo Fisher Scientific. Full uncropped immunoblot images are shown in Fig. S8.

B27 supplement was purchased from Life Technologies, Carlsbad, CA, USA (#17504-044), rhEGF from Sigma-Aldrich, St. Louis, MO, USA (#E9644), and recombinant human fibroblast growth factor (rhFGF) from BD Biosciences, San Jose, CA, USA (#354060).

Gelatin (Gel, Type A from porcine, 300 g bloom) was purchased from Sigma-Aldrich. Sodium alginate (Alg, low viscosity) was purchased from Alfa-Aesar. EDC and NHS were purchased from Beijing InnoChem Technology Co., Ltd. 1-hydroxybenzotrizole (HOBT, 98%) and carbohydrazide (CDH, 97%) were ordered from Heowns (Tianjin, China). Calcium chloride (CaCl₂) was purchased from Aladdin (Shanghai, China). rhEGF and TSA were purchased from Sigma-Aldrich. All the other reagents were of analytical grade and used without further purification.

To establish primary mammospheres, cells were plated on poly-HEMA coated dishes as single cell suspension in standard mammosphere medium containing phenol red-free DMEM/F12 (GIBCO), B27 supplement (50x, no vitamin A; Life Techonologies) and recombinant epidermal growth factor (rhEGF, 20 ng/ml; SIGMA). Where indicated, cells were plated in medium containing phenol red-free DMEM/F12, B27 supplement and 5% WF. In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium. After ten days, primary mammospheres were counted. To establish secondary mammospheres, primary mammospheres were collected, disaggregate in trypsin using 25-gauge needle fitted to a syringe. Cells were plated at the same seeding density of the primary generation. Mammosphere forming efficiency (MFE%) was calculated as follows: number of mammospheres per well/number of cells seeded per well × 100. Mammosphere self-renewal was calculated as follows: total number of secondary mammospheres /total number of primary mammospheres.

MDA-MB-231 were plated for 48 hours in complete medium or in serum free medium supplemented with recombinant epidermal growth factor (rhEGF, 20 ng/ml; SIGMA) or with 5% WF, in the presence or not of STAT3 inhibition. Cells were harvested, washed in PBS 1X and single-stained or double-stained with PE-CD24 (BD Biosciences #555428) and FITC-CD44 (BD Biosciences #347943) antibodies, for 20 minutes at RT. Cells were washed in PBS 1X and analyzed by flow cytometry (FACS LSR Fortessa, Becton Dickinson) to detect levels of surface markers CD44 and CD24.

MEDI3622 and IgG1 were directly obtained from AstraZeneca. Cells were pretreated with MEDI3622 or IgG1 24 hours prior to sham treatment or irradiation (5 Gy) with a RS-2000 225kV irradiator at 4.2 Gy/min (Rad Source).
The small molecular inhibitors TMI-005, SCH772984, and MG-132 were obtained from Axon Medchem (Axon 1507), Biovision (B1687) and Sigma (M7449), respectively. The human recombinant ligands rhEGF and rhAmphiregulin were obtained from Sigma Aldrich (E9644) and Genescript; (Z03103-50). rhEphrin-A1 Fc was obtained from R&D Systems (#6417-A1-050). Cells were treated as described in the results section. For sham treatment PBS or DMSO were used.