Dmem media

Colorectal cancer cell line, RKO, lymphoblast cell lines, TK6^TDP1+/+ and TK6^TDP1-/-, and prostate cancer cell line, LNCaP, were cultured in standard RPMI-1640 media (Lonza, USA). Transformed mouse embryonic fibroblasts MEF^TDP2+/+, MFE^TDP2-/-, and breast cancer cell line, MCF-7 were cultured in DMEM media (Lonza, USA). Both media were supplemented with 10% FBS (Sigma, USA) and 1% penicillin/streptomycin (Lonza, USA). The cells were maintained at 37 °C in 5% CO₂ incubator. For cellular synchronization double-thymidine block was used by treatment with 2 mM thymidine for 16 h, then released for 6 h in fresh media, and finally treated with thymidine for another 16 h. Hyperthermia treatment was applied by maintaining cells at 37, 43, or 45 °C in CO₂ incubators (for long term treatment) or water bath (short term treatment). All cell lines were obtained from the University of Sheffield (El-Khamisy lab) and routinely tested negative for mycoplasma.

Three cancer cell lines were screened for the anti-proliferative activity of the plant extract, namely human hepatocyte carcinoma (HEPG-2), human breast adenocarcinoma (MCF-7), and human colon carcinoma (HCT-116) cell lines. Quantitative measurement of the anti-cancer activity was performed using the protocol reported by Borenfreund and Puerner [76 (link)]. In this neutral red assay protocol, DMEM media (Lonza, Basel, Switzerland) was used to culture the cell lines which were supplemented with L-glutamine (0.2 M) and fetal bovine serum (10%), Gibco-BRL (Waltham, MA, USA). Dimethyl sulfoxide and DMEM mixture (at ratio 4/100, v/v) were used to dissolve the test compounds. The cell lines were tested using an initial dose of 1 mg/mL, which was followed by 7 serial dilutions for the dose (at 50% diluting factor) from the initial start dose. A concentration of 60,000 cells/mL of cells was seeded for 24 h in a 96-wells plate that was flat bottomed in conditions of 37 °C and carbon dioxide (5%) until obtaining a semi confluent cell layer. Then, the cell lines were treated with 100 µL of each dilution prepared serially of the test compounds. The anticancer activities were quantitatively assessed after 48 h using ELISA microplate readerset at 540 nm under the mentioned protocol [76 (link)].

Eight human colon cancer cell lines, SW1116, SW480, DLD-1, SW620, HT-29, Caco-2, COLO205 and T84 were obtained from the American Type Culture Collection (ATCC). SW1116, SW480, DLD-1, SW620, HT-29 and Caco-2 were cultured in DMEM media (Lonza) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). COLO205 was maintained in RPMI 1640 media (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin. T84 was maintained in DMEM/Ham's F12 media (Lonza) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. Cells were grown at 37°C in a humidified atmosphere of 5% CO₂.
Dukes' stage and tissue origin for each cell line is shown in Table S1.

MC38 and MB49 cell lines were cultured in DMEM media (Lonza) supplemented with 10% FCS (Hyclone), 2 mmol/L glutamine (Corning) and 100 U/mL penicillin/streptomycin (Corning) and passaged less than 8 times prior to inoculation. To establish the bilateral tumor model, 1 × 10⁶ MC38 cells were subcutaneously injected into the left or right flanks of mice. Once the size of the tumors reached 200 mm³, mice were randomized and received the following treatments: 1) vehicle control, 2) administration of 6-OAU for 10 days, 3) three doses of intraperitoneal (i.p.) injection with 200 μg anti-PD-1 (10F.9G2) every other day, or 4) combination of 6-OAU and α-PD-1 mAb. Tumor volumes were measured three times a week and calculated as [longest dimension × (perpendicular dimension²)]/2. Mice were euthanized when the tumor was greater than 2000 mm³. Mice were considered cured when tumor size was lower than 10 mm³.

Two different HCC cell lines were used in the study: Hep3B, with epithelial characteristics and moderate autocrine expression of TGF-β, and SNU449 cells, which show a mesenchymal phenotype and higher autocrine TGF-β expression [10 (link),11 (link)]. Both cell lines were obtained from the European Collection of Cell Cultures (ECACC) and were never used in the laboratory for longer than four months after receipt or resuscitation. Cells were maintained in DMEM media (Lonza, Basel, Switzerland) supplemented with 10% FBS (Sera Laboratories International Ltd., West Sussex, UK), penicillin (100 U/mL), streptomycin (100 μg/mL) and amphotericin (2.5 μg/mL) and L-glutamine (2 mM). They were maintained in a humidified atmosphere at 37 °C, 5% CO₂. For chronic TGF-β treatment, human recombinant TGF–β1 (Calbiochem, La Jolla, USA) was used at 2 ng/mL and replaced every 48 h. Stable transfection of the TGF-β Receptor I (TβRI) was performed as previously published [11 (link)]. Cells were observed under an Olympus 70iX microscope. Further information about targeted knock-down of TβRI and immunofluorescence analysis is included in Supplementary Materials and Methods .

U2OS cells (American Type Culture Collection, HTB-96) were cultured in DMEM media (Lonza, 12–604F) supplemented with 10% FBS (Thermo Fisher Scientific, A4766801) at 37°C and 5% CO₂ on coverslips. Cells were fixed by 2% paraformaldehyde (Electron Microscopy Sciences, 15711) in 1× PBS at room temperature for 15 minutes and rinsed three times with 1× PBS. Cells were incubated with Alexa Fluor 488 phalloidin (Invitrogen, A12379, 1:50 dilution in 1× PBS) for 1 hour at room temperature and rinsed three times with 1× PBS before imaging.

The B16-F10 tumor cell line was originally purchased from American Type Culture Collection (ATCC) and obtained from Dr. Susan Kaech (Salk Institute, San Diego, CA, USA) in 2013; it has not been further authenticated and tested for Mycoplasma. The B16-GP33 tumor cell line was generated as described [49 (link)] using the GP33-expressing plasmid generously provided by Hanspeter Pircher (University of Freiburg, Freiburg im Breisgau, Germany). Tumor cells were cultured in DMEM media (Lonza, Morristown, NJ, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 2 mmol/L L-glutamine (Corning, Corning, NY, USA), and 100 U/mL penicillin/streptomycin (Corning, Corning, NY, USA). Tumors were established by subcutaneous injection of 2 × 10⁵ tumor cells into the flanks of mice. Tumor growth was measured using a caliper and tumor volumes were calculated as [length × (width)²]/2 (The Jackson Laboratory, Bar Harbor, ME, USA).